SNP markers‐based map construction and genome‐wide linkage analysis in Brassica napus

An Illumina Infinium array comprising 5306 single nucleotide polymorphism (SNP) markers was used to genotype 175 individuals of a doubled haploid population derived from a cross between Skipton and Ag‐Spectrum, two Australian cultivars of rapeseed (Brassica napus L.). A genetic linkage map based on 613 SNP and 228 non‐SNP (DArT, SSR, SRAP and candidate gene markers) covering 2514.8 cM was constructed and further utilized to identify loci associated with flowering time and resistance to blackleg, a disease caused by the fungus Leptosphaeria maculans. Comparison between genetic map positions of SNP markers and the sequenced Brassica rapa (A) and Brassica oleracea (C) genome scaffolds showed several genomic rearrangements in the B. napus genome. A major locus controlling resistance to L. maculans was identified at both seedling and adult plant stages on chromosome A07. QTL analyses revealed that up to 40.2% of genetic variation for flowering time was accounted for by loci having quantitative effects. Comparative mapping showed Arabidopsis and Brassica flowering genes such as Phytochrome A/D, Flowering Locus C and agamous‐Like MADS box gene AGL1 map within marker intervals associated with flowering time in a DH population from Skipton/Ag‐Spectrum. Genomic regions associated with flowering time and resistance to L. maculans had several SNP markers mapped within 10 cM. Our results suggest that SNP markers will be suitable for various applications such as trait introgression, comparative mapping and high‐resolution mapping of loci in B. napus.     

A miR-1207-5p binding site polymorphism abolishes regulation of HBEGF and is associated with disease severity in CFHR5 nephropathy

Heparin binding epidermal growth factor (HBEGF) is expressed in podocytes and was shown to play a role in glomerular physiology. MicroRNA binding sites on the 3'UTR of HBEGF were predicted using miRWalk algorithm and followed by DNA sequencing in 103 patients diagnosed with mild or severe glomerulopathy. A single nucleotide polymorphism, miRSNP C1936T (rs13385), was identified at the 3'UTR of HBEGF that corresponds to the second base of the hsa-miR-1207-5p seed region. When AB8/13 undifferentiated podocytes were transfected with miRNA mimics of hsa-miR-1207-5p, the HBEGF protein levels were reduced by about 50%. A DNA fragment containing the miRSNP allele-1936C was cloned into the pMIR-Report Luciferase vector and co-transfected with miRNA mimics of hsa-miR-1207-5p into AB8/13 podocytes. In agreement with western blot data, this resulted in reduced luciferase expression demonstrating the ability of hsa-miR-1207-5p to directly regulate HBEGF expression. On the contrary, in the presence of the miRSNP 1936T allele, this regulation was abolished. Collectively, these results demonstrate that variant 1936T of this miRSNP prevents hsa-miR-1207-5p from down-regulating HBEGF in podocytes. We hypothesized that this variant has a functional role as a genetic modifier. To this end, we showed that in a cohort of 78 patients diagnosed with CFHR5 nephropathy (also known as C3-glomerulopathy), inheritance of miRSNP 1936T allele was significantly increased in the group demonstrating progression to chronic renal failure on long follow-up. No similar association was detected in a cohort of patients with thin basement membrane nephropathy. This is the first report associating a miRSNP as genetic modifier to a monogenic renal disorder.     

Silencing of nicotinamide nucleotide transhydrogenase impairs cellular redox homeostasis and energy metabolism in PC12 cells

Mitochondrial NADPH generation is largely dependent on the inner-membrane nicotinamide nucleotide transhydrogenase (NNT), which catalyzes the reduction of NADP(+) to NADPH utilizing the proton gradient as the driving force and NADH as the electron donor. Small interfering RNA (siRNA) silencing of NNT in PC12 cells results in decreased cellular NADPH levels, altered redox status of the cell in terms of decreased GSH/GSSG ratios and increased H(2)O(2) levels, thus leading to an increased redox potential (a more oxidized redox state). NNT knockdown results in a decrease of oxidative phosphorylation while anaerobic glycolysis levels remain unchanged. Decreased oxidative phosphorylation was associated with a) inhibition of mitochondrial pyruvate dehydrogenase (PDH) and succinyl-CoA:3-oxoacid CoA transferase (SCOT) activity; b) reduction of NADH availability, c) decline of mitochondrial membrane potential, and d) decrease of ATP levels. Moreover, the alteration of redox status actually precedes the impairment of mitochondrial bioenergetics. A possible mechanism could be that the activation of the redox-sensitive c-Jun N-terminal kinase (JNK) and its translocation to the mitochondrion leads to the inhibition of PDH (upon phosphorylation) and induction of intrinsic apoptosis, resulting in decreased cell viability. This study supports the notion that oxidized cellular redox state and decline in cellular bioenergetics - as a consequence of NNT knockdown - cannot be viewed as independent events, but rather as an interdependent relationship coordinated by the mitochondrial energy-redox axis. Disruption of electron flux from fuel substrates to redox components due to NNT suppression induces not only mitochondrial dysfunction but also cellular disorders through redox-sensitive signaling.     

Molecular Variability in North Indian Isolates of Cylindrocladium quinquieseptatum Causing Eucalyptus Leaf and Seedling Blight

Cylindrocladium quinqueseptatum has been considered as the most destructive pathogen of Eucalyptus nurseries and plantations in north India. Genetic resistance has not been determined against this disease in Eucalyptus and genetic diversity among the fungal population in northern India is not known. Seventy three isolates from infected leaves and twigs of Eucalyptus were collected from different northern Indian state and analyzed through RAPD-PCR for screening genetic diversity. The UPGMA cluster analysis score of 284 loci permitted identification of 11 population lines and an outlier. This molecular variability prevalent among the north Indian population of the pathogen can used in identifying Cylindrocladium leaf and seedling blight resistant Eucalyptus germplasm.     

Oxalate contributes to the resistance of Gaillardia grandiflora and Lupinus sericeus to a phytotoxin produced by Centaurea maculosa

Centaurea maculosa Lam. is a noxious weed in western North America that produces a phytotoxin, (±)-catechin, which is thought to contribute to its invasiveness. Areas invaded by C. maculosa often result in monocultures of the weed, however; in some areas, North American natives stand their ground against C. maculosa and show varying degrees of resistance to its phytotoxin. Two of these resistant native species, Lupinus sericeus Pursh and Gaillardia grandiflora Van Houtte, were found to secrete increased amounts of oxalate in response to catechin exposure. Mechanistically, we found that oxalate works exogenously by blocking generation of reactive oxygen species in susceptible plants and reducing oxidative damage generated in response to catechin. Furthermore, field experiments show that L. sericeus indirectly facilitates native grasses in grasslands invaded by C. maculosa, and this facilitation can be correlated with the presence of oxalate in soil. Addition of exogenous oxalate to native grasses and Arabidopsis thaliana (L.) Heynh grown in vitro alleviated the phytotoxic effects of catechin, supporting the field experiments and suggesting that root-secreted oxalate may also act as a chemical facilitator for plant species that do not secrete the compound.     

Cyanogenic pseudomonads influence multitrophic interactions in the rhizosphere

In the rhizosphere, plant roots cope with both pathogenic and beneficial bacterial interactions. The exometabolite production in certain bacterial species may regulate root growth and other root-microbe interactions in the rhizosphere. Here, we elucidated the role of cyanide production in pseudomonad virulence affecting plant root growth and other rhizospheric processes. Exposure of Arabidopsis thaliana Col-0 seedlings to both direct (with KCN) and indirect forms of cyanide from different pseudomonad strains caused significant inhibition of primary root growth. Further, we report that this growth inhibition was caused by the suppression of an auxin responsive gene, specifically at the root tip region by pseudomonad cyanogenesis. Additionally, pseudomonad cyanogenesis also affected other beneficial rhizospheric processes such as Bacillus subtilis colonization by biofilm formation on A. thaliana Col-0 roots. The effect of cyanogenesis on B. subtilis biofilm formation was further established by the down regulation of important B. subtilis biofilm operons epsA and yqxM. Our results show, the functional significance of pseudomonad cyanogenesis in regulating multitrophic rhizospheric interactions.     

Role of polyamines in the ontogeny of plants and their biotechnological applications

Recent developments in the metabolism and function of polyamines in plants is presented. Polyamines appear to be involved in a wide range of plant processes, however their exact role is not completely understood. In this review, the metabolic pathways involved in polyamine biosynthesis and degradation are explained, along with the transport and conjugation of these compounds. The studies involved in the understanding of function(s) of polyamines using metabolic inhibitors, as well as genetic and molecular approaches are described. Polyamine metabolism and profound changes in polyamine titres in response to infection by pathogens has been presented. Its role in adaptation of plants to stress is also presented. Molecular understanding of polyamines and their modulation in transgenics is also discussed. Further line of work in the understanding of the role of polyamines has also been focussed.     

Oncolytic virus therapy of multiple tumors in the brain requires suppression of innate and elicited antiviral responses.

The occurrence of multiple tumors in an organ heralds a rapidly fatal course. Although intravascular administration may deliver oncolytic viruses/vectors to each of these tumors, its efficiency is impeded by an antiviral activity present in complement-depleted plasma of rodents and humans. Here, this activity was shown to interact with complement in a calcium-dependent fashion, and antibody neutralization studies indicated preimmune IgM has a contributing role. Short-term exposure to cyclophosphamide (CPA) partially suppressed this activity in rodents and humans. At longer time points, cyclophosphamide also abrogated neutralizing antibody responses. Cyclophosphamide treatment of rats with large single or multiple intracerebral tumors substantially increased viral survival and propagation, leading to neoplastic regression.     

Focus Issue on Roots: Functional Soil Microbiome: Belowground Solutions to an Aboveground Problem

There is considerable evidence in the literature that beneficial rhizospheric microbes can alter plant morphology, enhance plant growth, and increase mineral content. Of late, there is a surge to understand the impact of the microbiome on plant health. Recent research shows the utilization of novel sequencing techniques to identify the microbiome in model systems such as Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). However, it is not known how the community of microbes identified may play a role to improve plant health and fitness. There are very few detailed studies with isolated beneficial microbes showing the importance of the functional microbiome in plant fitness and disease protection. Some recent work on the cultivated microbiome in rice (Oryza sativa) shows that a wide diversity of bacterial species is associated with the roots of field-grown rice plants. However, the biological significance and potential effects of the microbiome on the host plants are completely unknown. Work performed with isolated strains showed various genetic pathways that are involved in the recognition of host-specific factors that play roles in beneficial host-microbe interactions. The composition of the microbiome in plants is dynamic and controlled by multiple factors. In the case of the rhizosphere, temperature, pH, and the presence of chemical signals from bacteria, plants, and nematodes all shape the environment and influence which organisms will flourish. This provides a basis for plants and their microbiomes to selectively associate with one another. This Update addresses the importance of the functional microbiome to identify phenotypes that may provide a sustainable and effective strategy to increase crop yield and food security.In recent years, the term plant microbiome has received substantial attention, since it influences both plant health and productivity. The plant microbiome encompasses the diverse functional gene pool, originating from viruses, prokaryotes, and eukaryotes, associated with various habitats of a plant host. Such plant habitats range from the whole organism (individual plants) to specific organs (e.g. roots, leaves, shoots, flowers, and seeds, including zones of interaction between roots and the surrounding soil, the rhizosphere; Rout and Southworth, 2013). The rhizosphere is the region of the soil being continuously influenced by plant roots through the rhizodeposition of exudates, mucilages, and sloughed cells (Uren, 2001; Bais et al., 2006; Moe, 2013). Thus, plant roots can influence the surrounding soil and inhabiting organisms. Mutually, the rhizosphere organisms can influence the plant by producing regulatory compounds. Thus, the rhizospheric microbiome acts as a highly evolved external functional milieu for plants (for review, see Bais et al., 2006; Badri et al., 2009b; Pineda et al., 2010; Shi et al., 2011; Philippot et al., 2013; Spence and Bais, 2013; Turner et al., 2013a; Spence et al., 2014). In another sense, it is considered as a second genome to a plant (Berendsen et al., 2012). Plant rhizospheric microbiomes have positive or negative influence on plant growth and fitness. It is influenced directly by beneficial mutualistic microbes or pathogens and indirectly through decomposition, nutrient solubilization, nutrient cycling (Glick 1995), secretion of plant growth hormones (Narula et al., 2006; Ortíz-Castro et al., 2008; Ali et al., 2009; Mishra et al., 2009), antagonism of pathogens (Kloepper et al., 2004), and induction of the plant immune system (Pieterse et al., 2001; Ramamoorthy et al., 2001; Vessey, 2003; Rudrappa et al., 2008, 2010). The establishment of plant and rhizospheric microbiome interaction is a highly coordinated event influenced by the plant host and soil. Recent studies show that plant host and developmental stage has a significant influence on shaping the rhizospheric microbiome (Peiffer et al., 2013; Chaparro et al., 2014).There are various factors involved in the establishment of the rhizospheric and endophytic microbiome. They are greatly affected by soil and host type (Bulgarelli et al., 2012; Lundberg et al., 2012). Apart from these factors, other external factors such as biotic/abiotic stress, climatic conditions, and anthropogenic effects also can impact the microbial population dynamics in particular plant species. Plant host species differences can mainly be perceived from the secretory exudates by microbes. The root exudates act as a crucial driving force for multitrophic interactions in the rhizosphere involving microbes, neighboring plants, and nematodes (Bais et al., 2006). Thus, it is important to understand root exudate-shaped microbial community profiling in establishing phenotypes involved in plant health. Microbial components associated with plant hosts have to respond to these exudates along with utilizing them in order to grow competitively in a complex interactive root environment. Commonly, there are three groups of microbes present in the rhizosphere, commensal, beneficial, and pathogenic microbes, and their competition for plant nutrition and interactions confer the overall soil suppressiveness against pathogens and insects (Berendsen et al., 2012).Traditionally, the components of the plant microbiome were characterized by isolating and culturing microbes on different media and growth conditions. These culture-based techniques missed the vast majority of microbial diversity in an environment or in plant-associated habitats, which is now detectable by modern culture-independent molecular techniques for analyzing whole environmental metagenomes (comprising all organisms’ genomes). Over the last 5 years, these culture-independent techniques have dramatically changed our view of the microbial diversity in a particular environment, from which only less than 1% are culturable (Hugenholtz et al., 1998). After discovering the importance of the conserved 16S ribosomal RNA (rRNA) sequence (Woese and Fox, 1977) and the first use of denaturing gradient gel electrophoresis (DGGE) of the amplified 16S rRNA gene in the analysis of a microbial community (Muyzer et al., 1993), there was a sudden explosion of research toward microbial ecology using various molecular fingerprinting techniques. Apart from DGGE, thermal gradient gel electrophoresis, and fluorescence in situ hybridization, clone library construction of microbial community-amplified products and sequencing emerged as other supporting techniques for better understanding of microbial ecology (Muyzer, 1999). Furthermore, there are many newer techniques used to understand the microbiome, from metagenomics to metaproteomics (Friedrich, 2006; Mendes et al., 2011; Knief et al., 2012; Rincon-Florez et al., 2013; Schlaeppi et al., 2014; Yergeau et al., 2014). These techniques cover the whole microbiome, instead of selecting particular species, unlike conventional microbial analysis. However, their presence was not yet correlated well with the phenotypic manifestation (phenome) they establish in the host plant.As a consequence of population growth, food consumption is also increasing. On the other hand, cultivable agricultural land and productivity are significantly reduced due to global industrialization, drought, salinity, and global warming (Gamalero et al., 2009). This problem is only addressed by practicing the sustainable agriculture that protects the health of the ecosystem. The basic principle of sustainable agriculture is to significantly reduce the chemical input, such as fertilizers, insecticides, and herbicides, while reducing the emission of greenhouse gas. Manipulation of the plant microbiome has great potential in reducing the incidence of pests and diseases (van Loon et al., 1998; Kloepper et al., 2004; Van Oosten et al., 2008), promoting plant growth and plant fitness, and increasing productivity (Kloepper and Schroth 1978; Lugtenberg and Kamilova, 2009; Vessey, 2003). Single strains or mixed inoculum treatments induced resistance to multiple plant diseases (Jetiyanon and Kloepper, 2002). In recent years, several microbial biofertilizers and inoculants were formulated, produced, marketed, and successfully used by farmers worldwide (Bhardwaj et al., 2014). Although plants are being considered as a metaorganism (East, 2013), our understanding of the exact manifestation of this microbiome on plant health in terms of phenotypes is insufficient. Of late, there is a surge to understand and explore the genomic wealth of rhizosphere microbes. Hence, this Update will focus mainly on existing knowledge based on the root microbiome, its functional importance, and its potential relationship to the establishment of a host phenome, toward achieving sustainable agriculture.     

Draft Genome Sequences of Yersinia pestis Strains from the 1994 Plague Epidemic of Surat and 2002 Shimla Outbreak in India

We report the first draft genome sequences of the strains of plague-causing bacteria, Yersinia pestis, from India. These include two strains from the Surat epidemic (1994), one strain from the Shimla outbreak (2002) and one strain from the plague surveillance activity in the Deccan plateau region (1998). Genome size for all four strains is ~4.49 million bp with 139–147 contigs. Average sequencing depth for all four genomes was 21x.