Population Structure and Genetic Diversity in Popular Rice Varieties of India as Evidenced from SSR Analysis

We report here on the phylogenetic analysis, population substructure, and identification of molecular tags of 25 popular rice varieties and four landraces from different ecological belts of India employing a set of 52 simple sequence repeat (SSR) markers. Genetic analysis using the SSR markers categorized the genotypes into two major clusters, distributed according to their pedigree. Population structure analysis suggested that the optimum number of subpopulations was three (K?=?3) in the popular varieties and landraces. At K?=?5 the allelic distribution was much more similar to the phylogenetic dendrogram. The molecular diversity and population structure analysis indicated that there is not much variation among the popular rice cultivars of India. The study has identified SSR markers producing unique alleles, which should aid in the precise identification, maintenance, and genetic purity analysis of rice varieties.     

Comparative analysis of sequences of mitochondrial genomes of wild abortive male sterile (WA-CMS) and male fertile lines of rice,development of functional markers for WA-CMS trait and their use in assessment of genetic purity of seeds of WA-CMS lines

WA-CMS system based rice hybrids are widely adopted in many rice growing countries, including India. Even though it is well known that the trait is controlled by mitochondria, the genes underpinning the trait remain enigmatic. In the present study, a complete genome-wide comparative sequence analysis was performed using draft mitochondrial genomes of WA-CMS and male fertile lines in a step-wise manner, progressively covering 5–10 kb every time through BLASTN tool. The sequence polymorphisms identified in different mitochondrial regions were targeted to develop two different sets of dominant PCR-based markers, one consisting of six markers targeting WA-CMS mitochondria, the other set consisting of five markers targeting male fertile mitochondria in addition to development of a set of eight co-dominant PCR-based markers targeting both the genomes. When a set of candidate genes/ORFs reported earlier to be associated with WA-CMS trait in rice were analyzed through RT-PCR of RNA isolated from immature rice florets, it was observed that the chimeric ORF, WA352 is expressed only in WA-CMS line and hybrid (i.e. genotypes containing sterile mitochondria), indicating it’s candidacy for the WA-CMS trait. Targeting the functional nucleotide polymorphism between WA-CMS and maintainer mitochondria with respect to WA352, two dominant markers, one targeting sterile and another targeting fertile mitochondria were developed. In addition, a robust, co-dominant functional marker targeting the candidate gene was also developed and validated for its utility in identification of genetic impurities in seed lots of WA-CMS lines.     

QSAR modeling,pharmacophore-based virtual screening,and ensemble docking insights into predicting potential epigallocatechin gallate (EGCG) analogs against epidermal growth factor receptor

Epigallocatechin gallate (EGCG) is a major polyphenols of green tea may have the possibility to inhibit epidermal growth factor receptor (EGFR) activity and lead to reduce non-small cell lung cancer (NSCLC) progression. However, EGCG has some toxic features; moreover, there is a lack of explorations into the molecular interaction mechanisms of EGCG and the EGFR. In this examination, integration of quantitative structure–activity relationship (QSAR) modeling, pharmacophore-based virtual screening, and ensemble docking approaches were used to predict potential novel EGCG analogs as effective EGFR inhibitors. QSAR modeling of logP and logS predictions and toxicity endpoint investigation for a set of 82 compounds were shown good predictive ability and robustness from the applicability domain and confusion matrix elucidations. Virtual screening and docking studies revealed that seven high potential EGCG analogs as strong EGFR binders. Molecular interactions interpretations indicated some insights into the structural features of ligands that efficiently interfere with mutation possible residues (Gly⁷¹⁹ and Thr⁷⁹⁰) of the EGFR. The hydrogen bonds, hydrophobic interactions, atomic π-cation interactions and salt bridges of ligands are contributing additional stability to receptor structure, which can lead to blocking the intracellular protein-tyrosine kinase activity, including EGFR associated pathways activation in NSCLC. Therefore, this can characterize as a block-cluster mechanism between EGCG analogs and EGFR complexes. In silico anti-EGFR and anticancer activity predictions suggested that, ligands could act as promising pharmacological, anticancer, and drug-like templates of EGFR towards moderating the NSCLC progressions. These results and provided pinpoints could be beneficial to recognize probable therapeutic targets for NSCLC therapy.     

The bacteriophage, its role in immunology: how Macfarlane Burnet's phage research shaped his scientific style

The Australian scientist Frank Macfarlane Burnet-winner of the Nobel Prize in 1960 for his contributions to the understanding of immunological tolerance-is perhaps best recognized as one of the formulators of the clonal selection theory of antibody production, widely regarded as the 'central dogma' of modern immunology. His work in studies in animal virology, particularly the influenza virus, and rickettsial diseases is also well known. Somewhat less known and publicized is Burnet's research on bacteriophages, which he conducted in the first decade of his research career, immediately after completing medical school. For his part, Burnet made valuable contributions to the understanding of the nature of bacteriophages, a matter of considerable debate at the time he began his work. Reciprocally, it was while working on the phages that Burnet developed the scientific styles, the habits of mind and laboratory techniques and practices that characterized him for the rest of his career. Using evidence from Burnet's published work, as well as personal papers from the period he worked on the phages, this paper demonstrates the direct impact that his experiments with phages had on the development of his characteristic scientific style and approaches, which manifested themselves in his later career and theories, and especially in his thinking regarding various immunological problems.     

Isopentenyl pyrophosphate isomerase and prenyltransferase from tomato fruit plastids

Isopentenyl pyrophosphate isomerase has been isolated from an extract of tomato fruit plastids and purified 245-fold by fractionation with ammonium sulfate, gel filtration on Bio-Gel A 1.5m, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and chromatofocusing. Gel filtration on Sephadex G-100 separated the isopentenyl pyrophosphate isomerase from a prenyltransferase fraction that catalyzed the conversion of isopentenyl pyrophosphate to acid-labile compounds in the presence of dimethylallyl, geranyl, or farnesyl pyrophosphates. The molecular weights of the isopentenyl pyrophosphate isomerase and prenyltransferase were determined to be 34,000 and 64,000, respectively, by gel filtration on Sephadex G-100. The only cofactor required by either the isomerase or the prenyltransferase was a divalent cation, either Mg²⁺ or Mn²⁺. Isopentenyl pyrophosphate isomerase could also be totally inactivated by 1 × 10^?3m iodoacetamide, and this property was utilized in the assay of prenyltransferase activity in the presence of contaminating isomerase. The inactivation of isomerase by iodoacetamide is consistent with the stabilization of isopentenyl pyrophosphate isomerase by dithiothreitol. The K_m of isopentenyl pyrophosphate isomerase for isopentenyl pyrophosphate was found to be 5.7 × 10^?6.     

Purification and characterisation of prolactin from sheep and buffalo pituitaries

A study of the problem of structural variants of proteins and their relative contribution to the expressed immunological and biological activity has been initiated using sheep and buffalo prolactins as models. The feasibility of obtaining immunologically and biologically active prolactin in high yields from the discarded ’acid pellet’ of sheep and buffalo pituitaries has been demonstrated. This permits use of the same batch of glands for purifying lutropin, follitropin and prolactin as side fractions. The major component in preparations of buffalo prolactin has a molecular size of 24 kDa. The preparations were active in a radioligand binding inhibition assay and in a rat liver based radioreceptor assay. Charge and size isomers of sheep prolactin and buffalo prolactin have been observed. The reference sheep prolactin did not, in preliminary work, give any indication of being glycosylated. However radioactive sulphate was found to be incorporated into prolactin-rich fractions of sheep and buffalo pituitariesin vitro. By physico-chemical and immunochemical criteria the [³⁵S]-labelled material was similar to standard reference prolactin. The structural implications of sulphation have been probed. Presented at the 3rd National Symposium on Bioorganic Chemistry, 1987, Hyderabad.     

Subspecies — Specific Microsatellite Markers for Rice (Oryza sativa L)

Subspecific classification of Asian rice (Oryza sativa L) into indica and japonica has always been a subject of interest althrough for rice breeders and geneticists. The present study aims at identifying subspecies specific microsatellite markers in six genotypes, each of indica and japonica using 372 microsatellite primers covering the entire genome. Only 36 primers gave clear polymorphism on 3% agarose gel and these can be used as diagnostic markers for routine and easy identification of the subspecies.     

Ancestral sequence reconstruction in primate mitochondrial DNA: compositional bias and effect on functional inference

Reconstruction of ancestral DNA and amino acid sequences is an important means of inferring information about past evolutionary events. Such reconstructions suggest changes in molecular function and evolutionary processes over the course of evolution and are used to infer adaptation and convergence. Maximum likelihood (ML) is generally thought to provide relatively accurate reconstructed sequences compared to parsimony, but both methods lead to the inference of multiple directional changes in nucleotide frequencies in primate mitochondrial DNA (mtDNA). To better understand this surprising result, as well as to better understand how parsimony and ML differ, we constructed a series of computationally simple "conditional pathway" methods that differed in the number of substitutions allowed per site along each branch, and we also evaluated the entire Bayesian posterior frequency distribution of reconstructed ancestral states. We analyzed primate mitochondrial cytochrome b (Cyt-b) and cytochrome oxidase subunit I (COI) genes and found that ML reconstructs ancestral frequencies that are often more different from tip sequences than are parsimony reconstructions. In contrast, frequency reconstructions based on the posterior ensemble more closely resemble extant nucleotide frequencies. Simulations indicate that these differences in ancestral sequence inference are probably due to deterministic bias caused by high uncertainty in the optimization-based ancestral reconstruction methods (parsimony, ML, Bayesian maximum a posteriori). In contrast, ancestral nucleotide frequencies based on an average of the Bayesian set of credible ancestral sequences are much less biased. The methods involving simpler conditional pathway calculations have slightly reduced likelihood values compared to full likelihood calculations, but they can provide fairly unbiased nucleotide reconstructions and may be useful in more complex phylogenetic analyses than considered here due to their speed and flexibility. To determine whether biased reconstructions using optimization methods might affect inferences of functional properties, ancestral primate mitochondrial tRNA sequences were inferred and helix-forming propensities for conserved pairs were evaluated in silico. For ambiguously reconstructed nucleotides at sites with high base composition variability, ancestral tRNA sequences from Bayesian analyses were more compatible with canonical base pairing than were those inferred by other methods. Thus, nucleotide bias in reconstructed sequences apparently can lead to serious bias and inaccuracies in functional predictions.     

Quick and accurate detection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">carthami</Emphasis> in host tissue and soil using conventional and real-time PCR assay

Safflower wilt, caused by Fusarium oxysporum f. sp. carthami (Foc) is a major limiting factor for safflower (Carthamus tinctorius) production worldwide. In India alone, about 40–80% disease incidence has been reported. A rapid, efficient, specific, and sensitive diagnostic technique for Foc is therefore crucial to manage Fusarium wilt of safflower. Twenty-five isolates of F. oxysporum formae speciales infecting other crops, 17 isolates of Fusarium spp. and seven isolates of other fungal pathogens of safflower along with 75 Foc isolates were used for identification of band specific to Foc using inter-simple sequence repeat (ISSR) analysis. Out of 70 ISSR primers, the one that specifically amplified a 490 bp fragment from all the Foc isolates was selected. Sequence of the amplified fragment was utilized to design sequence characterized amplified region (SCAR) primers (FocScF/FocScR). The primer pair unambiguously and exclusively amplified a DNA fragment of approximately 213 bp in all the 75 Foc isolates. The primer set was able to detect as low as 10 pg of Foc genomic DNA using conventional PCR, while the SCAR primers when coupled with real-time qPCR demonstrated detection limits of 1 pg for Foc genomic DNA and 1000 conidia/g for soil. The assay enabled reliable diagnosis of Foc DNA in contaminated safflower fields and expedited Foc detection at 72 h post inoculation in asymptomatic seedlings. This method facilitates quick and precise detection of Foc in plant and soil samples and can be exploited for timely surveillance and sustainable management of the disease.