High-throughput multiplex microsatellite marker assay for detection and quantification of adulteration in Basmati rice (Oryza sativa)

Basmati rice is a very special type of aromatic rice known world-wide for its extra long grains and pleasant and distinct aroma. Traditional Basmati rice cultivars, confined to Indo-Gangetic regions of the Indian subcontinent, are often reported to be adulterated with crossbred Basmati varieties and long-grain non-Basmati varieties in the export market. At present, there is no commercial scale technology to reliably detect adulteration. We report here a CE-based multiplex microsatellite marker assay for detection as well as quantification of adulteration in Basmati rice samples. The single-tube assay multiplexes eight microsatellite loci to generate variety-specific allele profiles that can detect adulteration from 1% upwards. The protocol also incorporates a quantitative-competitive PCR-based analysis for quantification of adulteration. Accuracy of quantification has been shown to be ±1.5%. The experiments used to develop and validate the methodology are described

Novel female-specific splice form of dsx in the silkworm, Bombyx mori

The Bombyx mori doublesex (Bmdsx), a homologue of doublesex of Drosophila, is the bottom most gene of the sex determination cascade. Bmdsx plays a very crucial role in somatic sexual development. Its pre-mRNA sex-specifically splices to generate two splice variants; one encodes female-specific and the other encodes male-specific polypeptides which differ only at their C-termini. The open reading frame of Bmdsx consists of 5 exons, of which exons 3 and 4 are female-specific and are skipped in males. In the present study, we have identified a third splice form of the Bmdsx which is specific only to females and differs from the previously reported Bmdsxf isoform by the presence of 15 bp sequence. This new female splice form is generated as a result of alternative 5' splice site selection in the third exon adding additional 15 bp sequence in exon 3 which results in alteration of the reading frame leading to incorporation of an early stop codon. Thus the protein encoded by this splice form is 20 aa shorter than the known BmDsxF. Initial results obtained from the study of dsx homologues in Saturniid silkmoths suggest that both the female-specific Dsx proteins are essential for female sexual differentiation. It remains to be seen whether female-specific multiple splice forms of dsx are characteristic feature of only silkmoths or widespread among lepidopterans. The findings that sex determination mechanism is unique in lepidopterans offer an opportunity to develop genetic sexing methods in beneficial as well as economically destructive lepidopteran pests

Immune upregulation of novel antibacterial proteins from silkmoths (Lepidoptera) that resemble lysozymes but lack muramidase activity

Study on immune proteins in domesticated and wild silkmoths Bombyx mori and Antheraea mylitta, respectively, led to identification of a new class of antimicrobial proteins. We designated them as lysozyme-like proteins (LLPs) owing to their partial similarity with lysozymes. However, lack of characteristic catalytic amino acid residues essential for muramidase activity in LLPs puts them functionally apart from classical lysozymes. Two LLPs, one from B. mori (BLLP1) and the other from A. mylitta (ALLP1) expressed in a recombinant system, exhibited a broad-spectrum antibacterial action. Further investigation of the antibacterial mechanism revealed that BLLP1 is bacteriostatic rather than bactericidal against Escherichia coli and Micrococcus luteus. Substantial increase in hemolymph bacterial load was observed in B. mori upon RNA interference mediated in vivo knockdown of BLLP1. We demonstrate that the antibacterial mechanism of this protein depends on peptidoglycan binding unlike peptidoglycan hydrolysis or membrane permeabilization as observed with lysozymes and most other antimicrobial peptides. To our knowledge, this is the first report on functional analysis of novel, non-catalytic lysozyme-like family of antibacterial proteins that are quite apart functionally from classical lysozymes. The present analysis holds promise for functional annotation of similar proteins from other organisms

Genetic characterization of the Indian cattle breeds, Ongole and Deoni (Bos indicus), using microsatellite markers – a preliminary study

BACKGROUND: Molecular characterization of cattle breeds is important for the prevention of germplasm erosion by cross breeding. The Indian zebu cattle have their significant role in evolution of present day cattle breeds and development of some of the exotic breeds. Microsatellites are the best available molecular tools for characterization of cattle breeds. The present study was carried out to characterize two Indian cattle breeds, Ongole and Deoni, using microsatellite markers. RESULTS: Using 5 di- and 5 tri-nucleotide repeat loci, 17 Ongole and 13 Deoni unrelated individuals were studied. Of the ten loci, eight revealed polymorphism in both the breeds. The di-nucleotide repeat loci were found to be more polymorphic (100%) than tri-nucleotide repeat loci (60%). A total of 39 polymorphic alleles were obtained at 4.5 alleles per locus in Ongole and 4.1 in Deoni. The average expected heterozygosity was 0.46 (±0.1) and 0.50 (±0.1) in Ongole and Deoni breeds, respectively. The PIC values of the polymorphic loci ranged from 0.15 to 0.79 in Ongole and 0.13 to 0.80 in Deoni breeds. Six Ongole specific and three Deoni specific alleles were identified. The two breeds showed a moderate genetic relationship between themselves with a F(ST )value of 0.117 (P = 0.01). CONCLUSIONS: This preliminary study shows that microsatellite markers are useful in distinguishing the two zebu breeds namely, Ongole and Deoni. Further studies of other zebu breeds using many microsatellite loci with larger sample sizes can reveal the genetic relationships of Indian breeds

Multistep microsatellite mutation in the maternally transmitted locus D13S317: a case of maternal allele mismatch in the child

Examination of a case of a paternity dispute with 17 autosomal short tandem repeats (STR) loci revealed a mismatch of the maternally transmitted allele at the locus D13S317 in the questioned child. The composition of the alleles of this locus in the mother, questioned child and suspected father was 8/8, 11/11 and 8/11, respectively. The sequence analysis of the regions flanking the locus D13S317 and peak height measurements of the paternal, maternal and child alleles at this locus excluded the possibility of null allele as a cause of the allelic mismatch inherited by the child. The results suggested expansion of the microsatellite repeat motif, TATC by three repeat units as a probable cause for the allelic mismatch in the child. This is a rare case of maternally transmitted multistep microsatellite mutation reported for the first time for this locus in the forensic DNA analysis. The mutation rate at D13S317 locus in maternal and paternal meiosis was 0.04 and 0.14%, respectively, and overall mutation rate was 0.15%. The probability of maternity and paternity were 0.999999 and 0.999999, respectively, for all the 17 autosomal STR loci analyzed. Furthermore, the sequence of two hypervariable regions of mitochondrial DNA, HV1 and HV2 and the maternal alleles of six X chromosome STR loci in the questioned child matched completely with the mother. These results conclusively proved that the mother and suspected father are the biological parents of the questioned child

Genetic diversity and relationships in mulberry (genus Morus) as revealed by RAPD and ISSR marker assays

BACKGROUND: The genus Morus, known as mulberry, is a dioecious and cross-pollinating plant that is the sole food for the domesticated silkworm, Bombyx mori. Traditional methods using morphological traits for classification are largely unsuccessful in establishing the diversity and relationships among different mulberry species because of environmental influence on traits of interest. As a more robust alternative, PCR based marker assays including RAPD and ISSR were employed to study the genetic diversity and interrelationships among twelve domesticated and three wild mulberry species. RESULTS: RAPD analysis using 19 random primers generated 128 discrete markers ranging from 500–3000 bp in size. One-hundred-nineteen of these were polymorphic (92%), with an average of 6.26 markers per primer. Among these were a few putative species-specific amplification products which could be useful for germplasm classification and introgression studies. The ISSR analysis employed six anchored primers, 4 of which generated 93 polymorphic markers with an average of 23.25 markers per primer. Cluster analysis of RAPD and ISSR data using the WINBOOT package to calculate the Dice coefficient resulted into two clusters, one comprising polyploid wild species and the other with domesticated (mostly diploid) species. CONCLUSION: These results suggest that RAPD and ISSR markers are useful for mulberry genetic diversity analysis and germplasm characterization, and that putative species-specific markers may be obtained which can be converted to SCARs after further studies

A new Bombyx mori larval ovarian cell line highly susceptible to nucleopolyhedrovirus

Lepidopteran cell lines constitute the backbone for studying baculoviral biology in culturo and for baculovirus vector based recombinant protein expression systems. In the present study, we report establishment of a new continuous cell line designated as DZNU-Bm-1 from larval ovaries of the silkworm, Bombyx mori. The cells were grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum (FBS) and 3% heat inactivated B. mori haemolymph at 25 ± 1 °C. A large number of attached epithelial-like and round refractive cells migrated from the explants and multiplied in the primary cultures. Both type of cells were subcultured initially for a few passages but after 10 passages the round refractive cells dominated the population, which could be subcultured continuously using MGM-448 medium with 10% FBS. The population doubling time of cell line was about 42 h at 25 ± 1 °C. The cell populations were largely diploids and triploids, while a few tetraploids and hexaploids were also observed. DNA profiles using Inter Simple Sequence Repeat (ISSR)-PCR and Simple Sequence Repeat (SSR) loci established the differences between DZNU-Bm-1 cell line and most widely used BmN cell line and the B. mori W-chromosome specific sequences confirmed the origin of DZNU-Bm-1 cell line to be from female silkworm. When cells were infected with free nonoccluded B. mori nucleopolyhedrovirus (BmNPV), the cell line was found to be highly susceptible with 92-94% of the cells harbouring BmNPV and having an average of 20-23 OBs/infected cell. We suggest the usefulness of this cell line in BmNPV based baculoviral expression system and also for studying in culturo virus replication

Genetic diversity and population structure of Indian golden silkmoth (Antheraea assama)

Background The Indian golden saturniid silkmoth (Antheraea assama), popularly known as muga silkmoth, is a semi-domesticated silk producing insect confined to a narrow habitat range of the northeastern region of India. Owing to the prevailing socio-political problems, the muga silkworm habitats in the northeastern region have not been accessible hampering the phylogeography studies of this rare silkmoth. Recently, we have been successful in our attempt to collect muga cocoon samples, although to a limited extent, from their natural habitats. Out of 87 microsatellite markers developed previously for A. assama, 13 informative markers were employed to genotype 97 individuals from six populations and analyzed their population structure and genetic variation. Methodology/Principal Findings We observed highly significant genetic diversity in one of the populations (WWS-1, a population derived from West Garo Hills region of Meghalaya state). Further analysis with and without WWS-1 population revealed that dramatic genetic differentiation (global FST = 0.301) was due to high genetic diversity contributed by WWS-1 population. Analysis of the remaining five populations (excluding WWS-1) showed a marked reduction in the number of alleles at all the employed loci. Structure analysis showed the presence of only two clusters: one formed by WWS-1 population and the other included the remaining five populations, inferring that there is no significant genetic diversity within and between these five populations, and suggesting that these five populations are probably derived from a single population. Patterns of recent population bottlenecks were not evident in any of the six populations studied. Conclusions/Significance A. assama inhabiting the WWS-1 region revealed very high genetic diversity, and was genetically divergent from the five populations studied. The efforts should be continued to identify and study such populations from this region as well as other muga silkworm habitats. The information generated will be very useful in conservation of dwindling muga culture in Northeast India

Mechanism Involved in Biofilm Formation of <em>Enterococcus faecalis</em>

Enterococci are commensal bacteria in the gastrointestinal flora of animals and humans. These are an important global cause of nosocomial infections. A Biofilm formation constitutes an alternative lifestyle in which microorganisms adopt a multi-cellular behavior that facilitates and prolongs survival in diverse environmental niches. The species of enterococcus forms the biofilm on biotic and abiotic surfaces both in the environment and in the healthcare settings. The ability to form biofilms is among the prominent virulence properties of enterococcus. The present chapter highlights the mechanisms underlying in the biofilm formation by enterococcus species, which influences in causing development of the diseases

Genetic improvements to the sterile insect technique for agricultural pests

The sterile insect technique (SIT) relies on area-wide mass-releases of sterile male pest insects, which mate with their wild counterparts and thereby cause a drop in the wild population. In order to improve SIT efficacy or to avoid potential negative effects of such releases, strains of insects have been developed by genetic means. Methods of strain improvement fall into two categories: those generated by classical genetics and those through transgenesis. Here, we describe development and successes of agriculturally important pest insect strains developed through the former, and how transgenic technology is offering a broad spectrum of potential improvements to SIT in a wider range of insects. Also discussed are future prospects and non-technical challenges faced by transgenic technology. The need for environment-friendly pest control methods in agriculture has never been more pressing. SIT and related technologies offer a solution with proven effectiveness