PCR-based segregation of one hybrid variety of Labeo rohita and Catla catla from their wild-types

High consumer preference together with its polyculture potential has undoubtedly driven Rohu (Labeo rohita) and Catla (Catla catla) to top the list of the most preferred fishes among the Indian major carps. Commonly found in these fishes are hybrids that can be natural or man-made. Increasing emphasis on biodiversity issues has necessitated proper stock management of these through molecular genetics techniques. Also with few morphological differences that can be used to differentiate wild types and hybrids properly, the problem demands a straightforward molecular approach. Here, we report a simple PCR-based technique that can differentiate the hybrid variety from wild types easily using three different microsatellite markers. Three sets of primers were used to amplify three different microsatellite markers from the genomic DNA isolated from pectoral fins. When the PCR products using all three primer sets were analyzed, ‘hybrid–Rohu’ could be distinguished from wild types. Whereas the hybrid–Rohu DNA yielded specific PCR products with all three primer pairs, only two PCR products were obtained either from wild-type Catla DNA (by primer sets 1 and 2) or from wild-type Rohu DNA (by primer sets 1 and 3). This study clearly demonstrates that a simple PCR-based technique will help the fish breeders and hatcheries to identify and differentiate suspected hybrid–Rohu carp from the wild types within a few hours.     

Diversity Analysis in Selected Non-basmati Scented Rice Collection

Diversity analysis among 23 rice varieties including 16 non-basmati scented accessions, 5 basmati accessions and 2 non-scented accessions was performed by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) marker systems. The varieties analyzed by 11 RAPD and 8 ISSR primers yielded an average of 65% and 80% polymorphism, respectively. The average number of polymorphic bands generated per RAPD primer was 6 and per ISSR primer was 5.87. RAPD and ISSR data analysis individually could not segregate basmati and non-basmati scented rice accessions. However, the analysis using a combined data could group basmati and non-basmati scented rice accessions separately. The bands present specifically among three accessions of non-basmati scented rice were also identified. The study revealed a high genetic diversity among non-basmati scented rice accessions.     

Cross‐genera amplification of informative microsatellite markers from common bean and scarlet runner bean for assessment of genetic diversity in mungbean (Vigna radiata)

Mungbean (V. radiata) is an important Asiatic legume supplying inexpensive protein to a vast majority of vegetarian masses. To increase markers repertoire in mungbean, a study was conducted to analyse 384 microsatellite markers derived from common bean, scarlet runner bean and adzuki bean for their transferability and polymorphism. The results showed that 87 (24.71%) primer pairs could amplify DNA loci of 20 mungbean genotypes including one accession of V. trilobata, while 52 showed reliable banding and polymorphism. These showed different degrees of variability at each locus producing 250 alleles with the number of alleles varying from 2 to 9. The major allele frequency varied from 0.17 to 0.95, while the polymorphic information content of SSRs ranged between 0.09 and 0.86 with an average of 0.60 ± 0.16. UPGMA revealed three major clusters accommodating ~95% of the accessions while one accession of V. trilobata (‘NSB‐007’) did not group with any other genotype describing the discriminating power of informative microsatellites. This study identified a set of useful microsatellite markers to accelerate the genetic studies and breeding programme of mungbean.     

Genetic transformation of pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> L.) and screening transgenic progenies based on lateral root inhibition

Production of transgenic pigeonpea is becoming increasingly important, but the methods currently employed in production and subsequent screening still requires improvement. Here, we describe Agrobacterium-mediated genetic transformation of pigeonpea with reporter uidA (gus) gene and selectable marker, neomycin phospho-transferase (nptII) gene. Histochemical assay demonstrate localization of gus activity in cells and transformed plants. Overall, a transformation frequency of 0.33% was achieved using the protocol. Grafting of in vitro-regenerated healthy shoots indicates higher survival percent (72.6%), when stock and scion are of the same variety. Seeds harvested from primary transgenic plants can be screened based on lateral root inhibition strategy. Approximately 87% of the screened T₁ plants were found to be PCR positive. In conclusion, in vitro grafting of transgenic pigeonpea shoots leads to better plant establishment and screening based on lateral root inhibition leads to quick identification of positive segregants.     

Immunization of chickens with VP2 protein of infectious bursal disease virus expressed in Arabidopsis thaliana

Transgenic plants represent a safe, effective, and inexpensive way to produce vaccines. The immunogenicity of VP2 protein of an infectious bursal disease (IBD) virus variant E isolate expressed in transgenic Arabidopsis thaliana was compared with a commercial vaccine in specific-pathogen-free broiler chickens. The VP2 coding sequence was isolated and integrated into A. thaliana genome by Agrobacterium tumefaciens-mediated transformation. Soluble VP2 expressed in transgenic plants was used to immunize chickens. Chickens receiving oral immunization with plant-derived VP2 at 1 and 3 wk of age had an antibody response using enzyme-linked immunosorbent assay and 80% protection against challenge infection at 4 wk. Chickens primed with a commercial vaccine at 1 wk followed by an oral booster with VP2 expressed in plants at 3 wk of age showed 90% protection. Chickens immunized with a commercial vaccine at 1 and 3 wk showed 78% protection. Results supported the efficacy of plant-produced VP2 as a vaccine against IBD.     

A survey of gastro-intestinal parasitic infection in domestic and wild birds in Chittagong and Greater Sylhet,Bangladesh

A survey of gastrointestinal parasitic infection as determined by faecal examination was conducted among domestic and wild birds in Bangladesh. Birds were sampled from households, wet markets and wetlands in Chittagong and Greater Sylhet districts during April 2012 to February 2013. Mist nets were used to catch resident wild and migratory birds. The overall prevalence of parasitic infection ranged among locations from 25 to 55% in indigenous domestic ducks (live bird samples = 304), 20% in resident wild birds (environmental faecal samples = 40) and 40% in migratory birds (live bird samples = 35). The prevalence of parasitic infection was significantly higher in indigenous domestic ducks collected during summer (39%) than winter (22%) (p = 0.04). In domestic indigenous ducks and Muscovy ducks, both single and multiple types of parasitic infections were found. However, other domestic birds and wild birds often had a single type of parasitic infection. Ascaridia spp. with an average egg load of 50–900, was commonly detected in faecal samples of domestic and wild birds in this study. Other identified parasites were Capillaria spp. and Heterakis spp. both in domestic and wild birds. Improvement of biosecurity measures for household duck farms through educating and motivating household farmers could help mitigate the effects of parasitic infection on production.     

Allergenicity Assessment of Genetically-modified Tobacco Expressing Salt Tolerance cbl Gene

It is mandatory to assess the allergenic potential of genetically modified (GM) crops before their commercialization. Recently, a transgene [Calcineurin B-like (CBL) protein] has been introduced into tobacco plant to make the crop salt resistance. Therefore, it was felt necessary to assess the allergenic potential of the cbl gene product, which was introduced and expressed in Nicotiana tabacum (tobacco) plant and compared the allergenic effects with the wild-type (WT) counterpart. Bioinformatic analysis revealed that there was no significant sequence homology with known allergens. Also, no difference between the protein digestibility profiles of GM and WT tobacco was found. Rapid digestion of CBL protein (Mol Wt 35 kDa) by simulated gastric fluid (SGF) indicated reduced chances of this protein to induce allergenicity. In addition, BALB/c mice sensitized by intraperitoneal administration of WT and GM tobacco protein showed comparable levels of clinical score, specific IgE, IgG1, histamine level, similar effect on different organs as well as IgE binding proteins. These findings indicate that insertion of cbl gene in tobacco did not cause any additional allergic risk to consumer and the GM and native tobacco proteins behave similarly in both in vitro and in vivo situations even after genetic modification.