Evaluation of fluorescence in situ hybridization to detect encapsulated Bacillus pumilus SAFR-032 spores released from poly(methylmethacrylate)

Bacillus pumilus SAFR‐032 spores originally isolated from the Jet Propulsion Laboratory spacecraft assembly facility clean room are extremely resistant to UV radiation, H₂O₂, desiccation, chemical disinfection and starvation compared to spores of other Bacillus species. The resistance of B. pumilus SAFR‐032 spores to standard industrial clean room sterilization practices is not only a major concern for medical, pharmaceutical and food industries, but also a threat to the extraterrestrial environment during search for life via spacecraft. The objective of the present study was to investigate the potential of Alexa‐FISH (fluorescence in situ hybridization with Alexa Fluor® 488 labeled oligonucleotide) method as a molecular diagnostic tool for enumeration of multiple sterilant‐resistant B. pumilus SAFR‐032 spores artificially encapsulated in, and released via organic solvent from, a model polymeric material: poly(methylmethacrylate) (Lucite, Plexiglas). Plexiglas is used extensively in various aerospace applications and in medical, pharmaceutical and food industries. Alexa‐FISH signals were not detected from spores via standard methods for vegetative bacterial cells. Optimization of a spore permeabilization protocol capitalizing on the synergistic action of proteinase‐K, lysozyme, mutanolysin and Triton X‐100 facilitated efficient spore detection by Alexa‐FISH microscopy. Neither of the Alexa‐probes tested gave rise to considerable levels of Lucite‐ or solvent‐associated background autofluorescence, demonstrating the immense potential of Alexa‐FISH for rapid quantification of encapsulated B. pumilus SAFR‐032 spores released from poly(methylmethacrylate).     

SNPs in stress-responsive rice genes: validation, genotyping, functional relevance and population structure

ABSTRACT: BACKGROUND: Single nucleotide polymorphism (SNP) validation and large-scale genotyping are required to maximize the use of DNA sequence variation and determine the functional relevance of candidate genes for complex stress tolerance traits through genetic association in rice. We used the bead array platform-based Illumina GoldenGate assay to validate and genotype SNPs in a select set of stress-responsive genes to understand their functional relevance and study the population structure in rice. RESULTS: Of the 384 putative SNPs assayed, we successfully validated and genotyped 362 (94.3%). Of these 325 (84.6%) showed polymorphism among the 91 rice genotypes examined. Physical distribution, degree of allele sharing, admixtures and introgression, and amino acid replacement of SNPs in 263 abiotic and 62 biotic stress-responsive genes provided clues for identification and targeted mapping of trait-associated genomic regions. We assessed the functional and adaptive significance of validated SNPs in a set of contrasting drought tolerant upland and sensitive lowland rice genotypes by correlating their allelic variation with amino acid sequence alterations in catalytic domains and three-dimensional secondary protein structure encoded by stress-responsive genes. We found a strong genetic association among SNPs in the nine stress-responsive genes with upland and lowland ecological adaptation. Higher nucleotide diversity was observed in indica accessions compared with other rice sub-populations based on different population genetic parameters. The inferred ancestry of 16% among rice genotypes was derived from admixed populations with the maximum between upland aus and wild Oryza species. CONCLUSIONS: SNPs validated in biotic and abiotic stress-responsive rice genes can be used in association analyses to identify candidate genes and develop functional markers for stress tolerance in rice.     

Molecular Diversity in Indian Tobacco Types as Revealed by Randomly Amplified DNA Polymorphism

During the past five decades, a large number of tobacco varieties have been developed for different end uses in India through pure line selection from local land races, mutation breeding, and hybridization involving local selections and exotic introductions followed by pedigree selection. No systematic effort has been made to understand the existing diversity pattern in these varieties, which is crucial to define future breeding strategy in this important commercial crop. We characterized 46 varieties belonging to 10 different manufacturing tobacco types cultivated under different agro-climatic conditions in India along with two wild species of Nicotiana using 40 arbitrary primers in RAPID. The level of polymorphism among the varieties of N. tabacum was 59.4%, which was more than double the level observed in the other cultivated species N. rustica (25.2%). A broader range (0.64 to 0.94) of pair wise similarity measures in N. tabacum than in N. rustica (0.83 to 0.92) reflected the more diversified breeding efforts in the major cultivated species. The two wild species namely, N. glutinosa and N. gossei clustered separately from the two cultivated species. Molecular classification of the varieties corresponded largely with their manufacturing trait and parentage. RAPID markers provided sufficient resolution to distinguish among closely related tobacco types. Nine RAPID markers were found conserved across all the varieties and species. The markers found specific to the varieties can be used in correct identification of the carrier genotypes in trade and commerce. This is the first report on the molecular diversity analysis of Indian tobacco.     

Mapping of quantitative trait loci for basmati quality traits in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Traditional basmati rice varieties are very low yielding due to their poor harvest index, tendency to lodging and increasing susceptibility to foliar diseases; hence there is a need to develop new varieties combining the grain quality attributes of basmati with high yield potential to fill the demand gap. Genetic control of basmati grain and cooking quality traits is quite complex, but breeding work can be greatly facilitated by use of molecular markers tightly linked to these traits. A set of 209 recombinant inbred lines (RILs) developed from a cross between basmati quality variety Pusa 1121 and a contrasting quality breeding line Pusa 1342, were used to map the quantitative trait loci (QTLs) for seven important quality traits namely grain length (GL), grain breadth (GB), grain length to breadth ratio (LBR), cooked kernel elongation ratio (ELR), amylose content (AC), alkali spreading value (ASV) and aroma. A framework molecular linkage map was constructed using 110 polymorphic simple sequence repeat (SSR) markers distributed over the 12 rice chromosomes. A number of QTLs, including three for GL, two for GB, two for LBR, three for aroma and one each for ELR, AC and ASV were mapped on seven different chromosomes. While location of majority of these QTLs was consistent with the previous reports, one QTL for GL on chromosomes 1, and one QTL each for ELR and aroma on chromosomes 11 and 3, respectively, are being reported here for the first time. Contrary to the earlier reports of monogenic recessive inheritance, the aroma in Pusa 1121 is controlled by at least three genes located on chromosomes 3, 4 and 8, and similar to the reported association of badh2 gene with aroma QTL on chromosome 8, we identified location of badh1 gene in the aroma QTL interval on chromosome 4. A discontinuous 5 + 3 bp deletion in the seventh exon of badh2 gene, though present in all the RILs with high aroma, was not sufficient to impart this trait to the rice grains as many of the RILs possessing this deletion showed only mild or no aroma expression. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Detection and Mapping of Duplicate Loci in Brassica juncea

Restriction fragment length polymorphism (RFLP) analysis of Brassica juncea genome using 39 random homologous genomic DNA clones and chlorophyll a, b binding polypeptide (cab-3c) cDNA of tomato as probes revealed high degree of sequence duplication. The average number of hybridizing fragments per probe (8) was much higher than that earlier reported using cDNA probes in B. juncea. Null alleles observed for majority (56.2%) of the polymorphic duplicate loci suggested a significant role of insertion/deletion events in evolution of mustard genome. Distortion in segregation was evident in respect of only 9.6% of the segregating loci indicating that the mapping population used was relatively unbiased and thus can be used efficiently for genome mapping as well as for location of genes. Forty-nine polymorphic duplicate loci could be mapped to 15 linkage groups. Arrangement of these loci on different linkage groups revealed intra and inter-chromosomal duplications as well as duplication of chromosome blocks.Three of the eight cab loci could be mapped on three different linkage groups. Null allelic situation for seven of the cab loci suggested the role of DNA rearrangement in evolution of this multigene family in B. juncea.     

Evaluation of RAPD, ISSR and AFLP Markers for Characterization of the Loose Smut Fungus Ustilago tritici

Random Amplified Polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and Amplified Fragment Length Polymorphism (AFLP) profiling were evaluated for assessing the extent of genetic variation among the isolates of Ustilago tritici (Pers.) Rostr., which causes the loose smut disease of wheat.Thirty random decamer primers, six random primer pairs, four SSR primers such as (GACA)₄, (GATA)₄, (CAA)₅ and (GTG)₅ and nine combinations of AFLP selective primers were used to characterize nine isolates of the fungus. These isolates were collected from infected earheads of seven commercial wheat cultivars grown at eight different locations in Haryana, which is a major wheat growing state in the North-West Plain Zone of India. The RAPD and ISSR primers generated 21 0 scorable amplified fragments, all of which were monomorphic among the isolates.The AFLP primer combinations generated 239 fragments out of which 193 were polymorphic. All the isolates could be precisely differentiated from each other employing AFLP and grouped into two distinct clusters.The molecular classification partly corresponded with geographic distribution and host origin of the isolates. AFLP profiling was found superior to RAPD and ISSR and can be effectively utilized for further characterization of loose smut pathogen.     

Molecular Mapping of Loci Affecting the Contents of Three Major Fatty Acids in Indian Mustard (Brassica juncea L)

The fatty acid constituents of mustard oil are palmitic, stearic, oleic, linoleic, linolenic and erucic acids. With the objective of mapping loci influencing the content of these fatty acids, a population of F₆ generation recombinant inbred lines (RILs) derived from an inter-varietal cross of mustard was analyzed. Transgressive variation was evident for all the six fatty acids analysed irrespective of the levels of differences between the parents. The frequency distribution was normal for the linolenic acid, linoleic acid and stearic acid contents, while deviation from normality was observed for the other three fatty acids. The content of erucic acid was negatively correlated with the contents of all other fatty acids, which were positively correlated. Based on single marker analysis and interval mapping, two loci each for linoleic, linolenic and erucic acids were mapped to marker intervals on three linkage groups. Position of log of odds ratio (LOD) peaks suggested presence of common, linked and independently segregating loci for the fatty acid contents. The percentage of phenotypic variance explained by individual quantitative trait loci (QTLs) ranged from 10.5 to 19.5%, whereas the cumulative action of loci detected for different traits accounted for 16.3 to 27.6% of the variance. The additive effect for an individual locus ranged from 1.09 to 4.33. Presence of the favourable alleles at both the contributing loci in most of the RILs with a high linolenic acid content and of the unfavourable alleles in the lines with a low linolenic acid content indicated the possibility of pyramiding useful genes from phenotypically similar parental lines.     

Reduced Uptake as a Mechanism of Zinc Tolerance in Oscillatoria anguistissima

Oscillatoria anguistissima could tolerate 50 ppm ZnSO₄.7H₂O, and a zinc-tolerant strain with maximum tolerance concentration (MTC) of 100 ppm ZnSO₄.7H₂O was obtained by stepwise transfer to higher concentrations. The adaptation was irreversible even after three generations in metal-free medium. In the presence of metal, the tolerant strain grew with a shorter lag period of 4 days as against 6 days in the case of the wild strain. The tolerant strain had higher MTC than that of the wild strain for other metals also, viz., Ni²⁺, Co²⁺, Cu²⁺ and Cd²⁺. The zinc resistance in the tolerant strain was a result of reduced uptake, since around 42% of the total metal was present on the surface as against only 30% in the wild strain. The calcium-stimulated uptake, as observed in the wild strain, was absent in the tolerant strain. Ultrastructural comparisons revealed no structural change in the tolerant strain on exposure to zinc, whereas in the wild strain a thick extracellular matrix was observed. Received: 25 January 2001 / Accepted: 9 March 2001     

shRNAPred (version 1.0): An open source and standalone software for short hairpin RNA (shRNA) prediction

The small hairpin RNAs (shRNA) are useful in many ways like identification of trait specific molecular markers, gene silencing and characterization of a species. In public domain, hardly there exists any standalone software for shRNA prediction. Hence, a software shRNAPred (1.0) is proposed here to offer a user-friendly Command-line User Interface (CUI) to predict 'shRNA-like' regions from a large set of nucleotide sequences. The software is developed using PERL Version 5.12.5 taking into account the parameters such as stem and loop length combinations, specific loop sequence, GC content, melting temperature, position specific nucleotides, low complexity filter, etc. Each of the parameters is assigned with a specific score and based on which the software ranks the predicted shRNAs. The high scored shRNAs obtained from the software are depicted as potential shRNAs and provided to the user in the form of a text file. The proposed software also allows the user to customize certain parameters while predicting specific shRNAs of his interest. The shRNAPred (1.0) is open access software available for academic users. It can be downloaded freely along with user manual, example dataset and output for easy understanding and implementation. AVAILABILITY: The database is available for free at http://bioinformatics.iasri.res.in/EDA/downloads/shRNAPred_v1.0.exe.