Operon Primers Research Articles

Genomic variation in self-pollinated clove (Syzygium aromaticum (L.) Merr. & Perry) accessions from the Western Ghats

Clove, the dried, unopened flower buds of Syzygium aromaticum (L.) Merr. & Perry, is the mainstay of income for farmers in India, Indonesia, Tanzania and Sri Lanka and is a self-pollinated crop. A study conducted in the Southern Western Ghats of India identified phenotypic variations in the clustering of clove flower buds. Four clove accessions (Acc.1, Acc.3, Acc.5 and Acc.7) that showed superior yield and variation in floral characteristics were selected and DNA was isolated from the floral tissue using the CTAB method. Random Amplified Polymorphic DNA (RAPD) analysis using Operon primers (OPB 01 to OPB 10) detected polymorphism among the accessions, with OPB-01 showing the highest Polymorphic Information Content (PIC), followed by OPB-04 and OPB-06. Similarity coefficients were calculated based on the presence or absence of polymorphic bands, with coefficients ranging from 0.47 to 0.72. The resulting phylogenetic tree classified the accessions into two main groups based on their bud clustering habit (branching): branching (Acc. 1 and Acc. 5) and non branching (Acc. 3 and Acc. 7). Acc. 3 showed distinct separation from the other genotypes at a coefficient of 0.47, while Acc. 7 separated from Acc. 1 and Acc. 5 at a coefficient of 0.60. Acc. 5 appeared genetically distinct, as it showed separation from the other accessions based on RAPD analysis. The distinctiveness of Acc.5 was further confirmed through 18S rRNA sequencing, suggesting potential intra-species genomic variations among the four clove phenotypes and hinting at the possibility of cross pollination.

The effect of polyamines and silver thiosulphate on micropropagation of date palm followed by genetic stability assessment.

There are some limitations in date palm micropropagation. These include low multiplication efficiency, low rooting rate, and high mortality experienced by in vitro raised plantlets during laboratory to soil transfer. The objective of the study was to determine the effect of the polyamines and Silver Thiosulphate (STS) on the enhancement of shoot multiplication and genetic stability of in vitro cultures of date palm cultivar Quntar. Media supplemented with 75mg L-1 SPD in combination with 10 mg L-1 STS gave the highest percentage of callus producing buds (83.34%) and average bud formation (16.3) per jar. The addition of PUT and STS to the medium was most effective on root formation and the number of roots per shoot, where the best result, 91.67% and 6.37 roots per shoot, respectively, were obtained using 75 mg L-1 PUT and 10 mg L-1 STS, resulting in fast-growing plantlets during acclimatization phase, reaching 80% of plant survival. The genetic fidelity assessment of plants derived from micropropagation was confirmed by RAPD analysis. Four operon primers were used, and all of them showed amplified unambiguous (OPA02, OPC-04, OPD-07, and OPE-15). All generated bands were monomorphic and had no variation among the tissue culture-derived plants tested. Accordingly, these results indicate that adding polyamines and silver thiosulfate to the nutrient medium of date palm cv. Quntar was beneficial to improving shoot organogenesis, rooting, and production of genetically stable date palm plants.

RAPD Primers Selection for Genetic Variation Analysis of Banana Plant (Musa spp.)

Identification of genetic diversity using molecular markers is important for basic information for plant conservation. Banana is a fruit bearing plants that important for food sources in human life. This study aimed to determine the optimum conditions of PCR-RAPD reaction and RAPD primers that suitable to amplify DNA fragmens. DNA isolation was done using modified of CTAB and chloroform isoamil alcohol. The samples used was young leaves of nine banana cultivar plants. Optimation was done using variety of DNA and MgCl2 concentration. Eight primers produced by Operon Primer Technology were tested. The DNA genomic concentration obtained was in the range of 23,3 ng/µl – 70 ng/µl. The optimum conditions of PCR-RAPD of banana plants that produce clear band were 50 ng/µl DNA template, 3 mM MgCl2 with the number of thermal cycles was 40 x. There were six RAPD primers that successfully amplified DNA : OPA 02, OPA 04, OPB 12, OPD 20, OPH 01, and OPH 03. The primer OPA-04 had the lowest resolving power value (4,4) , while OPH 01 had the highest (11,3) resolving power.

Genetic variability of Artemia franciscana populations from different salterns of southeast coast of India

Abstract The brine shrimp Artemia franciscana consists of a complex of phenotypes within a population confined to various salterns in southeast coast of India. Studies are scarce for their occurrence based on morphology and morphometric traits, while seldom any on their genetic variability. Therefore, the present study investigates the genetic variability among the populations of Artemia franciscana samples collected from four different hypersaline habitats Kelambakkam (KBM), Vedaranyam (VDM), Tuticorin (TCN) and Nagarcoil (NGC) from southeast coast of India. Since the genetic variability could be anywhere in their genomes the study was analyzed using two different Random Amplified Polymorphic DNA (RAPD) markers such as (i) the Operon series (OPK) and (ii) Enterobacterial Repetitive Intergenic Consensus (ERIC). The OPK primers generated 367 fragments which indicated polymorphism of 85.29%, Nei's diversity index of 0.25 ± 0.16 and Shannon's index as 0.39 ± 0.22 among the population. Furthermore, ERIC-PCR generated 138 fragments with 20.83% of polymorphism while the Nei's diversity and Shannon's index was 0.06 ± 0.13 and 0.10 ± 0.20 respectively. Interestingly, operon primers of OPK3 and OPK17 revealed the presence of genetic variations among the populations, and the results were consistent with ERIC markers. Scatter plot and cluster analyses revealed the existence of two major groups in the population collected from the study sites; one with KBM and VDM, while other with TUT and NGC. Hence, the present investigation attributes the genetic variations in a panmictic population of A. franciscana which exist as two distinct groups.

Analisis Keragaman Genetik Dyera costulata (Miq) Hook.f. Berdasarkan Marka “Random Amplified Polymorphic DNA”

Dyera costulata (Miq.) Hook.f (Apocynaceae) is a large tree of the lowland tropical rain forest of Southeast Asia that occurs in Thailand, the Malay Peninsula and on the islands of Sumatera and Borneo. Its economic value was in its latex, used as gum chile in the manifacture of chewing gum. Nowadays the timber of this species is largely utilized for the manufacture of pencils and picture frames. The information on genetic diversity of the species is very limited. Hence studies were initiated and genetic diversity were estimated using Random Amplified Polymorphic (RAPD) markers in 47 accessions of Dyera costulata procured from different geographical regions of Jambi. Four selected Operon primers (10 mer) generated a total of 90 consistent amplification products ranging from 150 bp to 2.8 Kb. The cluster analysis showed that the 47 individuals were separated into one main cluster and one individual. The range of genetic dissimilarity value among samples was from 0.06 to 0.71, while genetic distance among populations was from 0.17 to 0.42. These values showed that those 47 accessions of D. costulata from Jambi was genetically originated from diverse population.

Genetic variability in different growth forms of Dendrocalamus strictus: Deogun revisited

BackgroundBamboo is among the important plants that help shape the socio-economic fabric of rural India. It provides employment, sustains business ventures, has medicinal applications and even helps in carbon sequestration. Out of 125 indigenous species, Dendrocalamus strictus (Roxb.) Nees occupies 53% of the area of bamboo in the country. Moreover, D. strictus may be used in afforestation of wastelands and rural development programmes due to its adaptability in wider landscapes. Dendrocalamus strictus has different growth forms based on edaphic factors and climatic conditions. DNA profiling was used to analyse the genetic diversity among the different growth forms of D. strictus present in three different locations of Uttarakhand.MethodsThe study area includes three locations, first, reserve forest of the Forest Research Institute (FRI), Dehradun; second, Shivpuri near Byasi, Rishikesh; and third, Chiriapur range (Haridwar district). A standard method was used to isolate DNA from young leaves from ten clumps of each growth form. Ten RAPD primers were screened for polymorphism from A and N operon primers and a standard PCR protocol was followed to amplify and visualise DNA bands. The data matrix was analysed and interpreted using statistical software and methods.ResultsThe cluster analysis, genetic structure parameters, moderate coefficient of gene differentiation and low gene flow value all indicated that these growth forms are genetically dissimilar and that geographic separation as well as physiological/flowering barriers has influenced these variations. These genetically different growth forms can be called ecotypes.ConclusionsSuch a study has not been attempted previously with bamboo and will help inform the conservation of the genetic pool of bamboo ecotypes. Seeds of these ecotypes are monocarpic in nature, which means that bamboo plants flower once in their lifetime, so they must be collected and multiplied (as plantations) in their respective habitats.

'Candidatus Phytoplasma wodyetiae', a new taxon associated with yellow decline disease of foxtail palm (Wodyetia bifurcata) in Malaysia.

Landscape-grown foxtail palm (Wodyetia bifurcata A. K. Irvine) trees displaying symptoms of severe foliar chlorosis, stunting, general decline and mortality reminiscent of coconut yellow decline disease were observed in Bangi, Malaysia, during 2012. DNA samples from foliage tissues of 15 symptomatic palms were analysed by employing a nested PCR assay primed by phytoplasma universal ribosomal RNA operon primer pairs, P1/P7 followed by R16F2n/R2. The assay yielded amplicons of a single band of 1.25 kb from DNA samples of 11 symptomatic palms. Results from cloning and sequence analysis of the PCR-amplified 16S rRNA gene segments revealed that, in three palms, three mutually distinct phytoplasmas comprising strains related to 'Candidatus Phytoplasma asteris' and 'Candidatus Phytoplasma cynodontis', as well as a novel phytoplasma, were present as triple infections. The 16S rRNA gene sequence derived from the novel phytoplasma shared less than 96 % nucleotide sequence identity with that of each previously describedspecies of the provisional genus 'Ca. Phytoplasma', justifying its recognition as the reference strain of a new taxon, 'Candidatus Phytoplasma wodyetiae'. Virtual RFLP profiles of the R16F2n/R2 portion of the 16S rRNA gene and the pattern similarity coefficient value (0.74) supported the delineation of 'Ca. Phytoplasma wodyetiae' as the sole representative subgroup A member of a new phytoplasma ribosomal group, 16SrXXXVI.

PENAMPILAN RANDOM AMPLIFIED POLYMORPHIC DNA PADA Azadirachta indica A. Juss DARI TAMAN NASIONAL BALURAN

Azadirachta indica A. Juss (Apocynaceae) is a large tree of the lowland tropical rain forest of Southeast Asia that occurs in Thailand, the Malay Peninsula, on the island of Java (East Java) and Lesser Sunda Islands. Its economic value was in its wood (timber), and as medicinal plant. The information on genetic diversity of the species is very limited. Hence studies were initiated and genetic diversity estimated using RAPD markers in 27 accessions of A.indica procured from three geographical regions of TN Baluran and Balai Litbang Kehutanan. Seven selected Operon primers (10 mer) generated a total of 133 consistent amplification products ranging from 132 bp to 5.6 Kb. The cluster analysis separated the 27 individuals into 2 clusters. The range of genetic dissimilarityvalue among samples was from 0.07 to 0.33, while genetic distance among populations was from 0.04 to 0.10. These values showed that A. indica from TN Baluran was not genetically diverse population. Key words: Azadirachta indica, genetic diversity, RAPD

Genotypic variations of ten Indian cultivars ofColocasia esculentavar.antiquoromSchott. evident by chromosomal and RAPD markers

Detailed karyotype, genome size and RAPD marker analysis were employed to assess genetic diversity in Taro (Colocasia esculenta var antiquorom Schott.). Karyotype analysis revealed genotype specific chromosomal characteristics and structural alterations in chromosome with variations of ploidy from 2n = 2x = 28 (cv. Mothan, cv. Muktakeshi, cv. Sree Kiran, cv. Sree Pallavi, cv. Sunajhili) to 3n = 3x = 42 (cv. Banky, cv. DP-25, cv. Duradin, cv. H-3, cv. Telia) in the genome. Highly significant variations in the genomic length, volume and total form (TF) % were noted at variety level. Total genomic chromosome length varied from 46.96μm in cv. Sree Kiran to 100.49μm in cv. Duradin. Total genomic chromosome volume varied from 18.22μm3 in cv. Sunajhili to 38.22μm3 in cv. Duradin. Total form percentage was varied from 24.94% in cv. Sree Kiran to 39.04% in cv. H-3 confirming near metacentric to metacentric chromosomes in the karyotype. Significant variations in the 4C DNA content noted among the cultivars that ranged from 7.24 pg in cv. Sree Kiran to 18.24 pg in cv. Duradin; accordingly, genome size varied from ~7095 to 17875 Mbp. High genome size in all the triplod varieties with 3x = 42 chromosomes could be due to the presence of extra set of chromosomes in the genome or high amount of repetitive DNA. The variation in the genome size at the variety level might be attributed to loss or addition of highly repetitive sequences in the genome. Amplification of genomic DNA in 10 genotypes using Operon primers yielded 230 amplified DNA fragments, ranging in size from 200 to 2500bp out of which 79 bands were polymorphic. A total of 8 unique RAPD bands were observed among 10 taro genotypes that revealed primer wise polymorphism ranged from 16.66 to 47.36% with an average polymorphic percentage of 34.34%. Whereas, among the cultivars the polymorphic percentage varied from 3.70% between cv. DR-25 & cv. Duradin and cv. Telia & cv. H-3 to 41.94% between cv. Mothan & cv. Muktakeshi. Genetic similarity based on Jaccard’s coefficient varied from 0.54 to 0.96, indicating wide genetic variability among the varieties based on RAPD markers. Similarity measures and cluster analysis generally reflected the expected trends in relationships of diploid and triplod taro varieties. Dendrogram obtained from the genetic distances among the varieties could be useful for breeders to choose the diverse parents for breeding programme aimed at varietal improvement.

Polymorphism in Calotropis procera: preliminary genetic variation in plants from different phytogeographical regions of Egypt

Calotropis procera (Aiton) W. T. Aiton. (family: Asclepiadaceae) is an important medicinal shrub distributed in the tropical and subtropical regions of the world. In Egypt, it is distributed throughout different phytogeographical regions. Genetic analyses were performed to study polymorphism of 18 individual genotypes collected from six different localities across Egypt (Aswan, New Valley, Cairo-Suez road, Feran, Nuweibaa, and Arish), and to elucidate their population genetic relationships. Five Operon primers successfully generated reproducible polymorphic products. RAPD profiles revealed high levels of polymorphism for the studied individual genotypes (93.45 %). Both number and size of the amplified products varied considerably with different primers and a sum of 100 polymorphic and 7 monomorphic bands were generated in all the individuals under study. A total of 24 unique bands were also identified. The combination of all polymorphic bands generated by all five primers was enough to discriminate between the six different localities. The dendrogram constructed revealed that Aswan (south Nile Valley) and Feran valley (South Sinai mountain area) to be separated in two distinct clusters, while the other four localities in a third cluster. Similarity indices for the genetic distances showed that the highest similarity was (76.03 %) between the genotypes from Cairo-Suez road and Nuweibaa regions, while the lowest was between Feran and Nuweibaa genotypes (63.87 %). The study confirmed the high polymorphic nature of the species and showed that genotypes of the Aswan locality have a highly similar gene pool to genotypes of most localities. South Sinai Feran locality seems to be of a different genetic background. Future studies on material collected from other phytogeographical regions of the world, as well as, using other molecular marker techniques, will contribute to understanding of the species polymorphism and its phytogeographical relationships across different regions of the world.

Fertility restoration in three CMS systems of eggplant by the Rf genes of each other's systems and their SCAR marker

Abstract Three pollen non-formation type cytoplasmic male sterility (CMS) systems of eggplant ( Solanum melongena L.) were developed in our previous study, through repeated backcrossing, by using wild Solanum species Solanum aethiopicum L. Aculeatum Group, Solanum anguivi Lam. and Solanum grandifolium C. V. Morton as the cytoplasm donor and eggplant ‘Uttara’ as the recurrent pollen parent. Each CMS system has male fertile (MF) line which carries the fertility restorer ( Rf ) genes for the pollen non-formation type sterility. In the present study, fertility restoration ability in three CMS systems of eggplant by the Rf genes of each other's systems was evaluated. The male sterile (MS) line of each CMS system was pollinated with the pollen of MF lines of other CMS systems as well as its own CMS system. Among the nine cross combinations, six groups were obtained from crossing MS lines with MF lines of other CMS systems and three groups were obtained from crossing MS lines with MF lines of their own CMS system. The segregation of MF and MS plants was fitted well with a 3:1 ratio in almost all cross combinations. The segregation patterns revealed that two independent dominant Rf genes restore fertility in each CMS system. Pollen stainability and in vitro pollen germination rate of the progenies obtained from different cross combinations were varied from 61.0 to 89.8% and 0.7 to 8.0%, respectively. Each Rf gene of three CMS systems restores fertility of other CMS systems as well as its own CMS system. Moreover, fertility recovery action in these three kinds of CMS is similar. DNA markers linked to Rf genes were screened by bulked segregant analysis (BSA) using random amplified polymorphic DNA (RAPD). A total of 420 primers were used, and more than 65% of the primers yielded a considerable number of RAPD fragments in both MF and MS bulks. Only one Operon primer OPAB10 gave a reproducible polymorphic band of 1.9 kbp (OPAB10 1900 ) between bulks and was identified as linked to the Rf genes. Additionally, the RAPD marker OPAB10 1900 was converted to sequenced characterized amplified region (SCAR) marker designated as SCAB10 1900 by nucleotide sequencing. Findings of the present study may give us a potential way to explore CMS and the corresponding Rf genes from wild Solanum resources for the breeding program of eggplant.

Genetic Analysis and Molecular Identification of Virulence in Xanthomonas oryzae pv. oryzae Isolates.

Bacterial leaf blight (BLB) of rice is a very destructive disease worldwide and is caused by Xanthomonas oryzae pv. oryzae (Xoo). The aim of the present study was to examine if the Xoo virulence pathotypes obtained using phenotypic pathotyping could be confirmed using molecular approach. After screening of 60 Operon primers with genomic DNA of two Xoo isolates (virulent pathotype, Vr, and mildly virulent pathotype, MVr), 12 Operon primers that gave reproducible and useful genetic information were selected and used to analyze 50 Xoo isolates from 7 West African countries. Genetic analysis revealed two major Xoo virulence genotypes (Mta and Mtb) with Mta having two subgroups (Mta1 and Mta2). Mta1 (Vr1) subgroup genotype has occurrence in six countries and Mta2 (Vr2) in three countries while Mtb genotype characterized mildly virulence (MVr) Xoo isolates present in five countries. The study revealed possible linkage and correlation between phenotypic pathotyping and molecular typing of Xoo virulence. Xoo virulence genotypes were known to exist within country and there was evidence of Xoo pathogen migration between countries. Durable resistance rice cultivars would need to overcome both Mta and Mtb Xoo virulence genotypes in order to survive after their deployment into different rice ecologies in West Africa.

English

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo), is a very destructive rice disease worldwide. The aim of the present study was to examine if the Xoo virulence pathotypes obtained using phenotypic pathotyping could be confirmed using molecular approach. After screening of 60 Operon primers with genomic DNA of two Xoo isolates (virulent pathotype, Vr and mildly virulent pathotype, MVr), 12 Operon primers that gave reproducible and useful genetic information were selected and used to analyze 50 Xoo isolates from 7 West African countries. Genetic analysis revealed two major Xoo virulence molecular type (Mt) which were Mta and Mtb with Mta having two subgroups (Mta1 and Mta2). Mta1 (Vr1) subgroup genotype has occurrence in six countries and Mta2 (Vr2) in three countries while Mtb genotype characterized mildly virulence (MVr) Xoo isolates present in five countries. The study revealed possible linkage and correlation between phenotypic pathotyping and molecular typing of Xoo virulence. Durable resistance rice cultivars would need to overcome both Mta and Mtb Xoo virulence genotypes in order to survive after their deployment into different rice ecologies in West Africa.   Key words: Bacterial leaf blight, Xanthomonas oryzae pv. oryzae (Xoo), Xoo virulence pathotype, molecular typing, genomic DNA, Operon primer, Xoo virulence genotype, Xoo pathogen migration, West Africa.

Floral Color Polymorphism and Reproductive Success in Annatto (Bixa orellana L.)

Floral color polymorphism of annatto (Bixa orellana L.) offers a wide range of colors that are maintained in the population by either pollinators or non pollinator agents of selection. In the present study, maintenance of different floral colors was analyzed in relation to reproductive success of Bixa orellana. The different floral petal colors (white, amaranth rose, petunia purple or cobalt violet) were determined from selected plants with reflectance spectrophotometry. Phenotypic measures of other floral traits, female reproductive success, seed set, seed output and seed weight also revealed variation between different floral morphs. Records on seed set varied significantly for different floral color morphs. Maximum fruit maturation (58 %) was observed in amaranth rose and least fruit maturation (25 %) in the white morph. Seed set data indicates pollinators’ preference for more intensely colored flowers. This preference may be due to ability of the pollinators to distinguish the morphs through differentially reduced sensitivity at the green wavelengths. In flowers which received fewer insect visits, polymorphism might be maintained by self fertilization. The color morphs showed differences in Random Amplified Polymorphic DNA (RAPD) profile indicating a genetic basis for floral color variation and consequent differences in seed set. Out of 88 bands generated with nine operon primers, 70 were polymorphic. The present study provides valuable information on the influence of petal color on maternal fitness in B. orellana.

Identification of RAPD Markers Associated with Morphological, Biochemical and Ionomic Characteristics in Indian Tomato Genotypes

Random Amplified Polymorphic DNA (RAPD) technique is one of the simplest marker techniques used in the differentiation of organisms. In the present study, 28 Indian tomato genotypes along with morphological, biochemical and ionomic characteristics were subjected to RAPD analysis. Ten random primers were selected from 100 operon primers which produced 88 reproducible bands; 89.8 % (79) of bands were polymorphic and 10.2 % (9) were monomorphic. The total number of markers identified by SMA in different parameters were plant height-06, fruit number-07, fruit cluster-03, fruit weight-03, pH-03, titratable acidity-03, TSS-03, total sugars-07, reducing sugars-06, non-reducing sugars-02, and lycopene-05. In case of macroelements, the total number of markers identified in SMA was P-03, K-05, Ca-06, Mg-03; for microelements, Mn-03, Fe-01, Co-06, Ni-06, Cu-04, Bo-06, Na-04, Mo-07, Cd-02 and Li-06. The total markers associated by SMRA showed the following. Plant height-06, fruit number-11, fruit cluster-06, fruit weight-17, pH-03, titratable acidity-04, TSS-04, total sugars-19, reducing sugars-20, non-reducing sugars-14, lycopene-19, P-13, K-18, Ca-03, Mg-03, Iron-13, Co-04, Ni-13, Cu-16, Zn-26, Bo-22, Na-21, Mo-20, Cd-18 and Li-12. The study suggested that the RAPD markers identified could be used in breeding elite tomato genotypes with good characteristics.

Assessment Population Genetic Structure and Diversity among Indian Snow Trout (S.richardsonii) Fish Species

In this study, the levels of genetic variability were estimated in S.richadsonii fish populations from three different locations viz., Chirapani stream (pop 1), Gola (pop 2) and KosiRiver (pop 3) by using OPY Operon primers and 10 primers were produced distinct and repeatable amplification as well polymorphic loci. The higher proportion of polymorphic loci was observed in Champawat population as compared to other two populations and with the overall % polymorphisms were 14.76. The OPY 01, OPY 04, OPY 11 and OPY 16 primers were found to be the population specific banding patterns among these three populations. The Nei's genetic diversity (h) was found to be highest in pop1 andpop2 with 0.24 and 0.2 in pop3. The total gene diversity (Ht) in the population was 0.2962, within sample gene diversity (Hs) of 0.2321 and the genetic differentiation (GST) among the populations was 0.2164. The largest genetic distance (0.1565) was obtained between pop1 and pop2, whereas the smallest genetic distance was between pop1 and pop3 (0.1058), followed by pop3 and pop2 (0.1385). Further it is suggested that the levels of genetic identity and diversity obtained was very low and might be due to the less number of polymorphic loci and migration rate.

Genetic and metabolic diversity in Stevia rebaudiana using RAPD and HPTLC analysis

Context: Stevia rebaudiana Bertoni (Asteraceae) is an important medicinal plant and is much used due to its zero calories sweetening property. Stevia leaves as well as its extracts and pure compounds are currently used in the preparation of several medicines, food products and neutraceuticals.Objective: To study the genetic and metabolic variability in S. rebaudiana among accessions of different geographical regions of India using random amplified polymorphic DNA (RAPD) markers and high-performance thin layer chromatography (HPTLC) analysis.Materials and methods: The RAPD analysis of Stevia rebaudiana (11 accessions) was carried out using 20 random operon primers. Dendrogram was constructed for cluster analysis based on the unweighted pair group method with arithmetic means (UPGMA) using Winboot. The HPTLC analysis of all samples was carried out on silica using acetone:ethyl acetate:water (5:4:1, v/v/v) for fingerprinting and quantification of stevioside and rebaudioside A at 360 nm after spraying with anisaldehyde sulphuric acid.Results: Ten out of 20 primers screened were found most informative; amplification products of the genotypes yielded a total of 87 scorable bands (67 polymorphic), whereas genetic similarity (GS) coefficient (0.01–0.08) and polymorphism (67.24–92.40%) showed huge variability. Similarly, HPTLC analysis showed large variation among different samples with respect to their presence or absence of metabolite and their concentration.Conclusion: Out of the 11 Stevia accessions, Delhi and Mohali varieties showed much relatedness with each other and were concluded to be the superior genotype in context to RAPD and HPTLC analysis. The information obtained here could be valuable for devising strategies for cultivating this medicinal plant.

Genetic Diversity Based on Morphological And Molecular Markers in Cashew (Anacardium occidentaleL.) Genotypes.

Study undertaken for genetic diversity of 11 cashew genotypes of Eastern Vidarbha zone are described on the basis of morphological and RAPD markers. Cluster I was consisting nine genotypes of EVZ while cluster II and III consist only one genotype i.e. GCN-7 and Vengurla-4 respectively. Average intra and inter cluster distances of studied genotypes showed ranged from 0.00 to 45.04 showing highest intra cluster distance D=45.04 which includes nine genotypes and maximum average inter cluster distance was observed between cluster I and III (88.92) and cluster I and II (85.57). Characterization of eleven genotypes of cashew is carried out by using RAPD marker. Out of 30 operon primers, 21 showed no amplification whereas 5 monomorphic and 4 polymorphic bands were used for the study. Out of 22 bands, 8 bands were monomorphic and 14 were polymorphic. Selected primers revealed 63.33 per cent polymorphism. The primer OPE-15 revealed maximum percent polymorphism (80.66%). The similarity coefficient range from 0.47 to 0.94 in eleven genotypes of cashew and average similarity coefficient was about 0.70. High coefficient of genetic similarity (0.94) was found in between GCN-12 and GCN-11.

‘Candidatus Phytoplasma convolvuli’, a new phytoplasma taxon associated with bindweed yellows in four European countries

Plants of Convolvulus arvensis exhibiting symptoms of undersized leaves, shoot proliferation and yellowing, collectively defined as bindweed yellows, were sampled in different regions of Europe and assessed for phytoplasma infection by PCR amplification using phytoplasma universal rRNA operon primer pairs. Positive results were obtained for all diseased plants. RFLP analysis of amplicons comprising the16S rRNA gene alone or the16S rRNA gene and 16-23S intergenic spacer region indicated that the detected phytoplasmas were distinguishable from all other previously described rRNA gene sequences. Analysis of 16S rRNA gene sequences derived from seven selected phytoplasma strains (BY-S57/11, BY-S62/11, BY-I1015, BY-I1016, BY-BH1, BY-BH2 and BY-G) showed that they were nearly identical (99.9-100% gene sequence similarity) but shared less than 97.5% similarity with comparable sequences of other phytoplasmas. Thus, BY phytoplasmas represent a new taxon whose closest relatives are stolbur phytoplasma strains and 'Candidatus Phytoplasma fragariae' with which they share 97.2% and 97.1% 16S rRNA gene sequence similarity, respectively. Phylogenetic analysis of 16S rRNA gene sequences confirmed that bindweed yellows phytoplasma strains collectively represent a distinct lineage within the phytoplasma clade and share a common ancestor with previously published or proposed 'Candidatus Phytoplasma' taxa within a major branch including aster yellows and stolbur phytoplasmas. On the basis of unique 16S rRNA gene sequences and biological properties that include a single host plant species and a geographical distribution limited to parts of Europe, the bindweed yellows (BY) phytoplasmas represent a coherent but discrete taxon, 'Candidatus Phytoplasma convolvuli', with strain BY-S57/11 (GenBank accession no. JN833705) as the reference strain.

RAPD-PCR and Outer Membrane Protein Characterization of Vibrio alginolyticus and Vibrio parahaemolyticus Isolated from Diseased shrimp

Vibrio alginolyticus and V. parahaemolyticus were characterized by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and profiling of outer membrane proteins (OMP) to the genetic level to enable easier identification of these microorganisms from diseased shrimp (Penaeus monodon) and adopt control measures. Of 20 tested Operon primers (OPC), only OPC5 and OPC18 were used for PCR amlification of 70 isolates representing three strains of each species. OPC5 amplified 1-8 bands with sizes ranging 0.42-2.95 Kb in V. alginolyticus, 5-10 fragments with sizes ranging 0.449-3.55 Kb in V. parahaemolyticus, and one unique common band of 1.34 Kb in both species. When OPC18 was used, one dark band (2.4 Kb) was common to all V. alginolyticus strains and another dark band (1.77 Kb) was common to two strains and the V. alginolyticus ATCC reference strain. One protein band (37.63 kDa) was common to both V. alginolyticus and V. parahaemolyticus and could be used as a diagnostic OMP marker for both species. The simpler and more rapid dot-ELISA and indirect ELISA methods confirmed identification of these pathogenic bacteria.