Syed Muhammad Saqlan Naqvi

Evidence of recombination in the Banana bunchy top virus genome

Viruses serve as good model for evolutionary studies, owing to their short generation times and small genomes. Banana bunchy top virus (BBTV) is a significant subject being multicomponent circular single stranded DNA virus. BBTV belongs to family Nanoviridae and contains DNA-R, -U3, -S, -M, -C, and -N as integral genomic components. Evolutionary studies have shown genetic re-assortment of components among its isolates and revealed a concerted type evolution in non-coding regions of its genome. The DNA U3 having been shown as the most diverse component in our previous studies, was subjected to sequencing from some Pakistani isolates for the first time. Sequence analysis revealed intergenomic recombination in DNA-U3 among the isolates of two sub-groups and a very rare intragenomic recombination in Pakistani BBTV population. This indicates that like other evolutionary processes including intergenomic recombination, intragenomic recombination among the genomic components of the same isolate may also have a significant contribution in the evolution of BBTV genome. Intragenomic recombination therefore appears to be a unique way to generate genetic diversity in the multicomponent ssDNA viruses.

Assessment of genetic variability among selected species of Apocynaceae

Family Apocynaceae is an economically important family grown as ornamental plants and many wild species have medicinal uses as well. The aim of the present study was to understand the level and pattern of genetic variability among the selected individuals of Apocynaceae. For this purpose, three species of different genera of Apocynaceae, Thevetia peruviana, Alstonia scholaris and Catharanthus roseus, were collected from Rawalpindi and Quaid-i-Azam University forest, Islamabad. To evaluate the level of polymorphism within the species and members of different species, randomly amplified polymorphic DNA (RAPD) markers were used. A series of OPC RAPD primers were used; only six primers of OPC series gave amplification. Highest genetic variation at interspecific and intraspecific levels was shown by OPC 9 and the lowest polymorphism was observed in OPC 4. The data was analyzed by using software Statistica 5.5. In total 105 monomorphic and 272 polymorphic bands were produced from all primers. Therefore, out of 322 amplified products, 26% were monomorphic and 68% were polymorphic. Low genetic diversification was observed both at intraspecific and interspecific level. At the molecular level Alstonia scholaris and Catharanthus roseus (subfamily Plumerioideae) appeared in a group and Thevetia peruviana (subfamily Rauvolfoideae) formed another group, confirming the classification based on morphological characters.

Comparative analysis of regulatory elements in different germin-like protein gene promoters

Germin and germin-like proteins (GLPs) the members of cupin superfamily of proteins, which are functionally most diverse proteins. Germin and GLPs have some unique features as they are highly resistant to proteases and to degradation by heat, high pH and detergents like Sodium dodecyl Sulphate (SDS). They are water soluble extracellular enzymatic protein that may also have Oxalate Oxidase (OxO), Superoxide dismutase (SOD) or ADP-glucose pyrophosphate or phosphodiestrase (AGPPase) activities. At the moment seven GLP gene promoter from different organisms have been studied and published. These all promoter sequences have been analyzed in this study. It was observed that these promoters have important regulatory elements, which are involved in various important functions. These elements have been compared on the basis of location, copy number, and distributed on positive and negative strands. It was also observed that some of these elements are common and remained conserved among all GLP promoters during evolution. Such regulatory elements are commonly observed in seed storage proteins, dehydration in response to light, senescence observed on exposure to dark and in elements specific for expression in pollen. Moreover, these common elements are reported to be expressed under environmental stresses (salt and pathogen attack) and to growth regulators.

Trichophyton verrucosum infection in livestock in the Chitral district of Pakistan

INTRODUCTION Trichophyton verrucosum belongs to the dermatophyte fungi, closely related organisms that cause skin infections in animals and humans. T. verrucosum infection has been reported in livestock and people in different countries from all continents. Human cases have been reported in different areas of Pakistan, but there is little information about the animal source of the fungus. METHODOLOGY Dermatological specimens collected in the Chitral district of Pakistan for a study on mange in livestock were retrospectively analyzed for the presence of T. verrucosum. In total, 5,873 animals (1,087 cows, 2,033 goats, and 2,753 sheep) were screened for evidence of dermatological lesions during two surveys performed in the summer and winter seasons. Skin scrapings collected from animals with lesions were analyzed by direct microscopic examination after digestion in sodium hydroxide and a real-time polymerase chain reaction (PCR) targeting pathogenic Trichophyton species. RESULTS At microscopy, samples from 18 cows (1.6%), 3 sheep (0.1%), and 4 goats (0.2%) were positive for fungal elements consistent with T. verrucosum. PCR confirmed the microscopy results. The prevalence was lower than that reported in other countries in intensive breeding farms. Results agree with the literature regarding factors affecting T. verrucosum diffusion, i.e., infection was more prevalent in cattle, especially in younger animals during the winter season. CONCLUSIONS This study reports, for the first time, the presence of T. verrucosum in livestock in Pakistan. A better knowledge of the animal role in the spread of this fungus may allow the adoption of more efficient control measures and prophylaxis.

Multiplex PCR assay for identification of commonly used disarmed Agrobacterium tumefaciens strains

The success of Agrobacterium mediated plant transformation depends to a certain extent on appropriate selection of the A. tumefaciens strain for a particular plant species. Many stages in a plant transformation procedure are prone to bacterial contamination with similar antibiotic resistance that may compromise the identity of the A. tumefaciens strain used, in turn adversely affecting success of a transformation experiment. Different primer sets were designed to exploit genetic differences among different strains of A. tumefaciens which are commonly used for plant genetic transformation, to identity confirmation as well as to distinguish them from one another. The primer sets Ach5FtsZ-F/R specific for Ach5 and C58GlyA-F/R specific for C58 were designed on chromosomal DNA while primer sets pTiBo542-F/R and nptI-F/R specific for plasmid pTiBo542 are capable to identify and distinguish these strains from one another. These primer sets when used simultaneously in multiplex PCR, produce a pattern which uniquely identifies all these strains and distinguishes them except for GV3101 and C58C1, which can further be distinguished from each other by rifampicin screening. The multiplex PCR assay and primers being reported here serve as a valuable tool in determining the identity of A. tumefaciens strains at any stage of plant transformation procedure.

Transgenic Analysis Reveals 5′ Abbreviated OsRGLP2 Promoter(s) as Responsive to Abiotic Stresses

Germins and germin-like proteins are ubiquitous, expressed at various developmental stages and in response to various abiotic and biotic stresses. In this study, to functionally validate the OsRGLP2 promoter, 5′ deletion analysis of the promoter sequences was performed and the deletion fragments fused with the β-glucuronidase (GUS) and green fluorescent protein reporter genes were used for transient expression in tobacco as well as for generating stable transgenic Arabidopsis plants. Very high level of GUS activity was observed in agroinfiltrated tobacco leaves by the construct carrying the P-1063 and P-565 when subjected to abiotic stresses. Histochemical analysis of transgenic Arabidopsis plants revealed expression of reporter gene in root, leaf and stem sections of plants harboring P-1063 and P-565. Real-time qPCR analysis of transiently expressed tobacco leaves and transgenic Arabidopsis plants subjected to several abiotic stresses supported histochemical data and showed that P-565 responded to all the stresses to which the full-length promoter was responsive. The data suggest that P-565 may be a good alternative to full-length promoter region that harbors the necessary cis-elements in providing stable and high level of expression in response to wound, salt and temperature stresses.

In vitro callogenesis and detection of somaclonal variations in Plantago ovata L.

Planago ovata L. is an economically important species in the monotypic genus Plantago. It is a short-stemmed annual herb. The seed husk of this plant is commonly called psyllium or isabgol which is important in pharmaceutical formulation and food industry. In this study, callus induction was optimized using different explants of Plantago ovata. Callus DNA was utilized to access the somaclonal variations using the Random Amplification of Polymorphic DNA (RAPD) markers. The maximum callus growth was observed in Murashige and Skoog (MS) medium containing 4 mg L−1 2,4-D concentration for shoots, 0.5 mg L−1 for seeds and 2 mg L−1 for roots. Moreover, the effect of culture age was considered in assessing genetic variability. Maximum genetic variability was observed in the DNA samples of callus at the concentration of 2 mg L−1 2,4-D for all explants (roots, shoots, and seeds). Cluster analysis was performed based on 1) similarity coefficient between samples and 2) molecular data using the Numerical Taxonomy and Multivariate Analysis System (NTSYS) PC version 2.01; similarity index was generated by similarity for Quantitative Data (SIMQUAL). Our study indicated that Random Amplified Polymorphic DNAs can successfully be used to explore polymorphism among callus samples at different hormonal concentrations. This study can be useful for the production of callus from Plantago ovata and estimation of genetic variations due to tissue culture conditions. Evaluation of genetic variations can display novel features and manipulate genetic bottlenecks in Plantago ovata. New genetic variations in somaclones can bring vital insight for plant improvement.

Enhanced Fusarium oxysporum f. sp. tuberosi Resistance in Transgenic Potato Expressing a Rice GLP Superoxide Dismutase Gene

Germin like proteins (GLPs) are a large group of related and ubiquitous plant proteins which are considered to be involved in different processes important for plant development and defense. Multiple functional copies of this gene family have been reported in a number of species (wheat, barley, rice, soybean mosses and liverwort), and their role is being evaluated by gene regulation studies and transgenic approaches. To analyze the role of a rice (Oryza sativa) root expressed germin like protein1 OsRGLP1, for its antifungal activity, transgenic potato plants were developed. These transgenic potato plants were molecularly characterized and biologically assessed after inoculation with Fusarium oxysporum f. sp. tuberosi. Functional analysis showed high accumulation of H2O2, increased Superoxide Dismutase (SOD) activity and no oxalate oxidase activity (OxO) in transgenics in comparison to nontransformed control. This increased SOD activity, resistance to heat and sensitivity to H2O2 suggest it is a Fe-like SOD. OsRGLP1 expression in potato plants exhibited enhanced resistance in comparison to nontransformed wild type plants suggesting its role in providing protection against Fusarium oxysporum f. sp. tuberosi through elevated SOD level. Overall, results suggest that OsRGLP1 is a candidate for the engineering of potato plants with increased fungal tolerance however, the greater height and tuber number was observed. This phenotype associated with the resistance needs to be evaluated to determine if this is a positive or negative feature.ResumenLas proteínas tipo germins (GLP) son un grupo grande de proteínas relacionadas de plantas y ubicuas, que se les considera que estan involucradas en diferentes procesos importantes para el desarrollo y defensa de la planta. Se han reportado múltiples copias funcionales de esta familia de genes en muchas especies (trigo, cebada, arroz, soya, musgos, plantas hepáticas), y su papel esta siendo evaluado mediante estudios de regulación génica y enfoques transgénicos. Para analizar la función de una proteína 1 tipo germins, OsRGLP1, expresada en la raíz de arroz (Oryza sativa), por su actividad antifúngica, se desarrollaron plantas de papa transgénicas. Éstas fueron caracterizadas molecularmente y evaluadas biológicamente después de la inoculación con Fusarium oxysporum f. sp. tuberosi. El análisis funcional mostró alta acumulación de H2O2, aumento de la actividad de la superóxido-dismutasa (SOD) e inactividad de la oxalato-oxidasa (OxO) en las transgénicas en comparación con las testigos no transformadas. Este aumento en la actividad de la SOD, resistencia al calor y sensibilidad a H2O2 sugiere que es una Fe-SOD. La expresión de OsRGLP1 en plantas de papa exhibió incremento en la resistencia en comparación con las plantas tipo silvestre no transformadas, lo que sugiere que su función es proporcionar protección contra Fusarium oxysporum f. sp. tuberosi mediante un nivel elevado de SOD. En general, los resultados sugieren que OsRGLP1 es una candidata para la ingeniería de plantas de papa con aumento de tolerancia a los hongos, no obstante, se observó la mayor altura y número de tubérculos. Este fenotipo asociado con la resistencia necesita evaluarse para determinar si esta es una característica positiva o negativa.