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Chickpea (Cicer arietinum L.) is the third most important cool season food legume, cultivated in arid and semi-arid regions of the world. The goal of this study was to develop novel molecular markers such as microsatellite or simple sequence repeat (SSR) markers from bacterial artificial chromosome (BAC)-end sequences (BESs) and diversity arrays technology (DArT) markers, and to construct a high-density genetic map based on recombinant inbred line (RIL) population ICC 4958 (C. arietinum)×PI 489777 (C. reticulatum). A BAC-library comprising 55,680 clones was constructed and 46,270 BESs were generated. Mining of these BESs provided 6,845 SSRs, and primer pairs were designed for 1,344 SSRs. In parallel, DArT arrays with ca. 15,000 clones were developed, and 5,397 clones were found polymorphic among 94 genotypes tested. Screening of newly developed BES-SSR markers and DArT arrays on the parental genotypes of the RIL mapping population showed polymorphism with 253 BES-SSR markers and 675 DArT markers. Segregation data obtained for these polymorphic markers and 494 markers data compiled from published reports or collaborators were used for constructing the genetic map. As a result, a comprehensive genetic map comprising 1,291 markers on eight linkage groups (LGs) spanning a total of 845.56 cM distance was developed (http://cmap.icrisat.ac.in/cmap/sm/cp/thudi/). The number of markers per linkage group ranged from 68 (LG 8) to 218 (LG 3) with an average inter-marker distance of 0.65 cM. While the developed resource of molecular markers will be useful for genetic diversity, genetic mapping and molecular breeding applications, the comprehensive genetic map with integrated BES-SSR markers will facilitate its anchoring to the physical map (under construction) to accelerate map-based cloning of genes in chickpea and comparative genome evolution studies in legumes.
We established a protocol of the SuperSAGE technology combined with next-generation sequencing, coined “High-Throughput (HT-) SuperSAGE”. SuperSAGE is a method of digital gene expression profiling that allows isolation of 26-bp tag fragments from expressed transcripts. In the present protocol, index (barcode) sequences are employed to discriminate tags from different samples. Such barcodes allow researchers to analyze digital tags from transcriptomes of many samples in a single sequencing run by simply pooling the libraries. Here, we demonstrated that HT-SuperSAGE provided highly sensitive, reproducible and accurate digital gene expression data. By increasing throughput for analysis in HT-SuperSAGE, various applications are foreseen and several examples are provided in the present study, including analyses of laser-microdissected cells, biological replicates and tag extraction using different anchoring enzymes.
Symbiotic nitrogen fixation (SNF) in root nodules of grain legumes such as chickpea is a highly complex process that drastically affects the gene expression patterns of both the prokaryotic as well as eukaryotic interacting cells. A successfully established symbiotic relationship requires mutual signaling mechanisms and a continuous adaptation of the metabolism of the involved cells to varying environmental conditions. Although some of these processes are well understood today many of the molecular mechanisms underlying SNF, especially in chickpea, remain unclear. Here, we reannotated our previously published transcriptome data generated by deepSuperSAGE (Serial Analysis of Gene Expression) to the recently published draft genome of chickpea to assess the root- and nodule-specific transcriptomes of the eukaryotic host cells. The identified gene expression patterns comprise up to 71 significantly differentially expressed genes and the expression of twenty of these was validated by quantitative real-time PCR with the tissues from five independent biological replicates. Many of the differentially expressed transcripts were found to encode proteins implicated in sugar metabolism, antioxidant defense as well as biotic and abiotic stress responses of the host cells, and some of them were already known to contribute to SNF in other legumes. The differentially expressed genes identified in this study represent candidates that can be used for further characterization of the complex molecular mechanisms underlying SNF in chickpea.
One of the most challenging aspects of RT-qPCR data analysis is the identification of reliable reference genes. Ideally, they should be neither induced nor repressed under different experimental conditions. To date, few reference genes have been adequately studied for sugarcane (Saccharum spp.) using statistical approaches. In this work, six candidate genes (αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were tested for gene expression normalization of sugarcane root tissues from drought-tolerant and -sensitive accessions after continuous dehydration (24 h). By undergoing different approaches (GeNorm, NormFinder, and BestKeeper), it was shown that most of them could be used in combinations for normalization purposes, with the exception of SAMDC. Nevertheless three of them (H1, αTUB, and GAPDH) were considered the most reliable reference genes. Their suitability as reference genes validated the expression profiles of two targets (AS and PFPα1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis. The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way. In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches.
Alternative polyadenylation (APA) is a widespread mechanism that contributes to the sophisticated dynamics of gene regulation. Approximately 50% of all protein-coding human genes harbor multiple polyadenylation (PA) sites; their selective and combinatorial use gives rise to transcript variants with differing length of their 3' untranslated region (3'UTR). Shortened variants escape UTR-mediated regulation by microRNAs (miRNAs), especially in cancer, where global 3'UTR shortening accelerates disease progression, dedifferentiation and proliferation. Here we present APADB, a database of vertebrate PA sites determined by 3' end sequencing, using massive analysis of complementary DNA ends. APADB provides (A)PA sites for coding and non-coding transcripts of human, mouse and chicken genes. For human and mouse, several tissue types, including different cancer specimens, are available. APADB records the loss of predicted miRNA binding sites and visualizes next-generation sequencing reads that support each PA site in a genome browser. The database tables can either be browsed according to organism and tissue or alternatively searched for a gene of interest. APADB is the largest database of APA in human, chicken and mouse. The stored information provides experimental evidence for thousands of PA sites and APA events. APADB combines 3' end sequencing data with prediction algorithms of miRNA binding sites, allowing to further improve prediction algorithms. Current databases lack correct information about 3'UTR lengths, especially for chicken, and APADB provides necessary information to close this gap. Database URL: http://tools.genxpro.net/apadb/
Chromatin, RNA Polymerase, Potato Tuber Tissue, Aging Phenomenon The synthesis of RNA by chromatin-bound RNA polymerase (E.C. 2.7.7.6.) from white potato tubers proceeds at a low rate, which is enhanced after slicing the tissue, however. Concomitantly DNA template availability as measured with saturating amounts of Escherichia coli polymerase is diminished drastically. Nearest neighbor frequency analysis proved that the RNA synthesized on chromatin of intact tubers is different from that synthesized on chromatin of sliced tissue.
The RNA polymerase of white potato tubers is dependent on all four ribonucleoside triphos phates and a divalent metal ion such as Mg2+ or Mn2+ and totally inhibited by the presence of pyrophosphate. Actinomycin D blocks the formation of the RNA product, which could be shown to be a heteropolymer by nearest neighbour frequency technique. The Km of the chromatin-bound enzyme with regard to ATP, GTP, CTP and UTP was 5.1 X10-5 M, 1.6X10-5 M, 0.9X10-5 M and 0.45 X 10-5M/1 respectively, α-amanitin inhibits the overall activity to about 50%, which indicates the presence of equal amounts of polymerase I and polymerase If.
Resting potato tuber tissue possesses only faint activity of the two dehydrogenases of the oxidative pentose phosphate cycle, glucose-6-phosphate- and 6-phosphogluconate dehydrogenase. Slicing of the tissue, however, greatly enhances the action of both enzymes. The slicing-induced increase in activity is a consequence of intensified action of at least 5 glucose-6-phosphate dehydrogenase isozymes and a more differentiated activation/inactivation of seven 6-phosphogluconate dehydrogenase isozymes.
Using density labelling and isopycnic equilibrium centrifugation it could be demonstrated, that the bulk of both enzymes appearing after slicing the tissue is the result of de novo synthesis rather than activation of pre-existing proenzymes.
In addition to the importance of many Dioscorea species (yams) as starchy staple food, some representatives are known and still used as a source for the steroidal sapogenin diosgenin, which, besides phytosterols derived from tall-oil, is an important precursor for partial synthesis of steroids for pharmaceutical research and applications. While in edible yams the diosgenin content should be as low as possible, a high yield of the compound is preferable for cultivars which are grown for the extraction of sterols. In the past, miscalculations and insufficiently precise techniques for quantification of diosgenin prevailed. Therefore we set out to re-evaluate the steroid content of a world collection of Dioscorea species, using leaves as sample material. We optimized diosgenin quantification techniques and fingerprinted the whole collection with the DNA amplification fingerprinting (DAF) technique. Total diosgenin contents ranged from 0.04 to 0.93% of dry weight within the collection. Several Dioscorea cultivars can be characterized via their DAF fingerprint patterns.