Development of genic-SSR markers by deep transcriptome sequencing in pigeonpea [Cajanus cajan (L.) Millspaugh]

<p>Abstract</p> <p>Background</p> <p>Pigeonpea [<it>Cajanus cajan </it>(L.) Millspaugh], one of the most important food legumes of semi-arid tropical and subtropical regions, has limited genomic resources, particularly expressed sequence based (genic) markers. We report a comprehensive set of validated genic simple sequence repeat (SSR) markers using deep transcriptome sequencing, and its application in genetic diversity analysis and mapping.</p> <p>Results</p> <p>In this study, 43,324 transcriptome shotgun assembly unigene contigs were assembled from 1.696 million 454 GS-FLX sequence reads of separate pooled cDNA libraries prepared from leaf, root, stem and immature seed of two pigeonpea varieties, Asha and UPAS 120. A total of 3,771 genic-SSR loci, excluding homopolymeric and compound repeats, were identified; of which 2,877 PCR primer pairs were designed for marker development. Dinucleotide was the most common repeat motif with a frequency of 60.41%, followed by tri- (34.52%), hexa- (2.62%), tetra- (1.67%) and pentanucleotide (0.76%) repeat motifs. Primers were synthesized and tested for 772 of these loci with repeat lengths of ≥18 bp. Of these, 550 markers were validated for consistent amplification in eight diverse pigeonpea varieties; 71 were found to be polymorphic on agarose gel electrophoresis. Genetic diversity analysis was done on 22 pigeonpea varieties and eight wild species using 20 highly polymorphic genic-SSR markers. The number of alleles at these loci ranged from 4-10 and the polymorphism information content values ranged from 0.46 to 0.72. Neighbor-joining dendrogram showed distinct separation of the different groups of pigeonpea cultivars and wild species. Deep transcriptome sequencing of the two parental lines helped <it>in silico </it>identification of polymorphic genic-SSR loci to facilitate the rapid development of an intra-species reference genetic map, a subset of which was validated for expected allelic segregation in the reference mapping population.</p> <p>Conclusion</p> <p>We developed 550 validated genic-SSR markers in pigeonpea using deep transcriptome sequencing. From these, 20 highly polymorphic markers were used to evaluate the genetic relationship among species of the genus <it>Cajanus</it>. A comprehensive set of genic-SSR markers was developed as an important genomic resource for diversity analysis and genetic mapping in pigeonpea.</p

Harnessing VLSI System Design with EDA Tools

This book explores various dimensions of EDA technologies for achieving different goals in VLSI system design. Although the scope of EDA is very broad and comprises diversified hardware and software tools to accomplish different phases of VLSI system design, such as design, layout, simulation, testability, prototyping and implementation, this book focuses only on demystifying the code, a.k.a. firmware development and its implementation with FPGAs. Since there are a variety of languages for system design, this book covers various issues related to VHDL, Verilog and System C synergized with EDA tools, using a variety of case studies such as testability, verification and power consumption. * Covers aspects of VHDL, Verilog and Handel C in one text; * Enables designers to judge the appropriateness of each EDA tool for relevant applications; * Omits discussion of design platforms and focuses on design case studies; * Uses design case studies from diversified application domains such as network on chip, hospital on chip, analog to digital conversion and embedded system design; * Facilitates with code and tool flows the design cycle for systems on chip with increasing complexity; * Demonstrates standard development cycles, making use of latest concepts such as ‘Soft IP Cores’, ‘Hardware Software Codesign’ etc

Harnessing VLSI System Design with EDA Tools

XXVIII, 156 p.online resource

Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics

Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute significantly to the observed T cell responses

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Not AvailableMango (Mangifera indica L.) is known as the ‘king of fruits’ for its rich taste, flavor, color, production volume and diverse end usage. It belongs to plant family Anacardiaceae and has a small genome size of 439 Mb (2n = 40). Ancient literature indicates origin of cultivated mango in India. Although wild species of genus Mangifera are distributed throughout South and South-East Asia, recovery of Paleocene mango leaf fossils near Damalgiri, West Garo Hills, Meghalaya point to the origin of genus in peninsular India before joining of the Indian and Asian continental plates. India produces more than fifty percent of the world’s mango and grows more than thousand varieties. Despite its huge economic significance genomic resources for mango are limited and genetics of useful horticultural traits are poorly understood. Here we present a brief account of our recent efforts to generate genomic resources for mango and its use in the analysis of genetic diversity and population structure of mango cultivars. Sequencing of leaf RNA from mango cultivars ‘Neelam’, ‘Dashehari’ and their hybrid ‘Amrapali’ revealed substantially higher level of heterozygosity in ‘Amrapali’ over its parents and helped develop genic simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. Sequencing of double digested restriction-site-associated genomic DNA (ddRAD) of 84 diverse mango cultivars identified 1.67 million high quality SNPs and two major sub-populations. We have assembled 323 Mb of the highly heterozygous ‘Amrapali’ genome using long sequence reads of PacBio single molecule real time (SMRT) sequencing chemistry and predicted 43,247 protein coding genes. We identified in the mango genome 122,332 SSR loci and developed 8,451 Type1 SSR and 835 HSSR markers for high level of polymorphism. Among the published genomes, mango showed highest similarity with sweet orange (Citrus sinensis). These genomic resources will fast track the mango varietal improvement for high productivity, disease resistance and superior end use qualityNot Availabl