Development and evaluation of high‐density Axiom®CicerSNP Array for high‐resolution genetic mapping and breeding applications in chickpea

To accelerate genomics research and molecular breeding applications in chickpea, a high‐throughput SNP genotyping platform ‘Axiom^®CicerSNP Array’ has been designed, developed and validated. Screening of whole‐genome resequencing data from 429 chickpea lines identified 4.9 million SNPs, from which a subset of 70 463 high‐quality nonredundant SNPs was selected using different stringent filter criteria. This was further narrowed down to 61 174 SNPs based on p‐convert score ≥0.3, of which 50 590 SNPs could be tiled on array. Among these tiled SNPs, a total of 11 245 SNPs (22.23%) were from the coding regions of 3673 different genes. The developed Axiom^®CicerSNP Array was used for genotyping two recombinant inbred line populations, namely ICCRIL03 (ICC 4958 × ICC 1882) and ICCRIL04 (ICC 283 × ICC 8261). Genotyping data reflected high success and polymorphic rate, with 15 140 (29.93%; ICCRIL03) and 20 018 (39.57%; ICCRIL04) polymorphic SNPs. High‐density genetic maps comprising 13 679 SNPs spanning 1033.67 cM and 7769 SNPs spanning 1076.35 cM were developed for ICCRIL03 and ICCRIL04 populations, respectively. QTL analysis using multilocation, multiseason phenotyping data on these RILs identified 70 (ICCRIL03) and 120 (ICCRIL04) main‐effect QTLs on genetic map. Higher precision and potential of this array is expected to advance chickpea genetics and breeding applications.     

RV-Typer: A Web Server for Typing of Rhinoviruses Using Alignment-Free Approach

Rhinoviruses (RV) are increasingly being reported to cause mild to severe infections of respiratory tract in humans. RV are antigenically the most diverse species of the genus Enterovirus and family Picornaviridae. There are three species of RV (RV-A, -B and -C), with 80, 32 and 55 serotypes/types, respectively. Antigenic variation is the main limiting factor for development of a cross-protective vaccine against RV.Serotyping of Rhinoviruses is carried out using cross-neutralization assays in cell culture. However, these assays become laborious and time-consuming for the large number of strains. Alternatively, serotyping of RV is carried out by alignment-based phylogeny of both protein and nucleotide sequences of VP1. However, serotyping of RV based on alignment-based phylogeny is a multi-step process, which needs to be repeated every time a new isolate is sequenced. In view of the growing need for serotyping of RV, an alignment-free method based on “return time distribution” (RTD) of amino acid residues in VP1 protein has been developed and implemented in the form of a web server titled RV-Typer. RV-Typer accepts nucleotide or protein sequences as an input and computes return times of di-peptides (k = 2) to assign serotypes. The RV-Typer performs with 100% sensitivity and specificity. It is significantly faster than alignment-based methods. The web server is available at http://bioinfo.net.in/RV-Typer/home.html.     

Non-linear pattern of radiation induced lipid peroxidation is not affected by vitamin E, Fe2+ ions and molecular oxygen.

Results of the present study on liposomes have clearly shown that non-linear pattern of radiation-induced lipid peroxidation was not changed even in the presence of vitamin E, Fe2+ ions or molecular oxygen. These results are important from biological point of view as lipid peroxidation is used as a measure of membrane damage.     

Proteins encoded by the DpnI restriction gene cassette. Hyperproduction and characterization of the DpnI endonuclease

Insertion mutations in the DpnI gene cassette of Streptococcus pneumoniae indicated that the two genes it contains, dpnC and dpnD, were transcribed from an adjacent promoter and that only dpnC was necessary for expression of the DpnI endonuclease. Large amounts of the DpnI endonuclease were produced from the cloned cassette in an Escherichia coli expression system, and the enzyme was purified to homogeneity. The DpnI endonuclease is composed of a single polypeptide of 30 kDa, which, as shown by NH2-terminal sequencing of the protein, is encoded by the entire dpnC open reading frame. The native protein sedimented as a monomer of 30 kDa in 0.5 M NaCl. A protein composed of a 20-kDa polypeptide, which is presumably encoded by dpnD, was also produced in large amounts. It was partially purified, but its function is unknown. Examination of the predicted amino acid sequence of DpnI revealed a potential metal-containing, DNA-binding finger structure. It is suggested that this structure provides the specificity for recognition of the methylated DNA sequence, 5'-GmATC-3', that is cleaved by the DpnI endonuclease.     

Apoptotic mechanisms during competition of ribosomal protein mutant cells: roles of the initiator caspases Dronc and Dream/Strica

Heterozygosity for mutations in ribosomal protein genes frequently leads to a dominant phenotype of retarded growth and small adult bristles in Drosophila (the Minute phenotype). Cells with Minute genotypes are subject to cell competition, characterized by their selective apoptosis and removal in mosaic tissues that contain wild-type cells. Competitive apoptosis was found to depend on the pro-apoptotic reaper, grim and head involution defective genes but was independent of p53. Rp/+ cells are protected by anti-apoptotic baculovirus p35 expression but lacked the usual hallmarks of ‘undead'' cells. They lacked Dronc activity, and neither expression of dominant-negative Dronc nor dronc knockdown by dsRNA prevented competitive apoptosis, which also continued in dronc null mutant cells or in the absence of the initiator caspases dredd and dream/strica. Only simultaneous knockdown of dronc and dream/strica by dsRNA was sufficient to protect Rp/+ cells from competition. By contrast, Rp/Rp cells were also protected by baculovirus p35, but Rp/Rp death was dronc-dependent, and undead Rp/Rp cells exhibited typical dronc-dependent expression of Wingless. Independence of p53 and unusual dependence on Dream/Strica distinguish competitive cell death from noncompetitive apoptosis of Rp/Rp cells and from many other examples of cell death.In Drosophila, heterozygous mutation of many ribosomal protein gene loci leads to the dominant ‘Minute'' phenotype, named for its small thin bristles.^{1, 2} Minute animals show a dominant developmental delay. In addition, Minute (that is, Rp/+) cells tend to be lost from mosaics that contain wild-type cells, making it difficult for clones of Rp/+ genotypes to survive and contribute to the adult.^{3, 4, 5, 6, 7} Such conditional cell viability that depends on a heterotypic cellular environment is termed ‘cell competition''.⁴Competition of Rp/+ clones is suppressed by equalizing growth rates through starvation⁸ or nonmosaic mutation of a second Rp locus.⁴ Hyperplastic clones that express higher levels of myc^{9, 10} or lower levels of the Salvador-Hippo-Warts pathway tumor suppressors out-compete nearby wild-type cells, that is, they are ‘super-competitors''.^{7, 11} Competition based on c-myc also occurs in mouse embryogenesis.¹² Differential growth is not always sufficient to cause cell competition, as cells growing rapidly due to elevated CyclinD/Cdk4 activity or higher activity of the insulin/IGF pathway are not super-competitive.⁹ Differences in Jak/Stat signaling, Wg signaling and cell adhesion are also reported to generate cell competition.^{13, 14, 15} These findings suggest that cell competition arises from specific interactions between cells, rather than as a general consequence of differential growth.Apoptotic cell death is a fundamental part of cell competition. Elimination of Rp/+ clones is delayed by expression of the caspase inhibitor baculovirus p35.⁵ Apoptosis of Rp/+ cells also occurs when clones of wild-type cells arise in Rp/+ backgrounds, predominantly among Rp/+ cells nearby wild-type cells.^{6, 16} As expected, such apoptosis is prevented by expression of baculovirus p35 or DIAP1.^{6, 16, 17}Cell competition has been hypothesized to contribute to human cancer, because most tumors have an altered genotype, and because many genes implicated in cell competition are homologs of oncogenes and tumor suppressors.^{18, 19, 20, 21} Cell competition may contribute to homeostasis of organ growth^{4, 9} and to antitumor surveillance.^{22, 23, 24, 25, 26}Cell competition may be a means to eliminate certain categories of aneuploid cells.^{27, 28} Seventy-nine ribosomal protein genes, sixty-six of which are haploinsufficient Minute loci, are distributed throughout the Drosophila genome.² Copy number changes to parts of the genome are likely to perturb relative dose of Rp/+ genes, and those that reduce Rp gene dose could be subject to cell competition. This suggests cell competition can eliminate some aneuploid cells even after DNA damage responses have ceased.^{27, 28, 29}In humans, heterozygosity for multiple different Rp mutations causes Diamond Blackfan Anemia.³⁰ Accumulation of ribosomal assembly intermediates or of unassembled ribosomal proteins in these genotypes activates p53, for example through the binding of the p53 ubiquitin ligase Mdm2 by RpL11 or RpL5.³¹ The p53 pathway leads to cell cycle arrest and/or apoptosis,³² and loss of hematopoietic stem cells causes anemia. Diamond Blackfan Anemia is a condition of nonmosaic individuals, so its relationship to cell competition is unclear.The uncertain nature of the cell interactions that trigger competition might be illuminated if the initiation of competitive apoptosis was understood. The Drosophila genome encodes three potential initiator caspases that might be activated through long prodomains, and four effector caspase zymogens lacking prodomains that are activated by initiator caspases and by one another.³³ Here, the p53 and initiator caspase requirements for competitive cell death of Rp/+ cells were determined. Whereas Dronc is the initiator caspase for most apoptosis in Drosophila,^{34, 35, 36, 37} we found that competitive cell death could occur without dronc or p53. Experiments that eliminated multiple initiator caspases simultaneously demonstrated that competitive apoptosis of Rp/+ cells required Dronc and Dream/Strica redundantly, a difference from most other apoptotic genotypes in Drosophila, for example, Rp/Rp cells generated in these experiments died in a Dronc-dependent manner.