Search results for: genome duplication

Authors: Anyaphat Srithanasuwan, Noppason Pangprasit, Montira Intanon, Phongsakorn Chuammitri, Witaya Suriyasathaporn, Ynte H. Schukken

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Streptococcus uberis is one of the most common mastitis-causing pathogens, with a wide range of intramammary infection (IMI) durations and pathogenicity. This study aimed to compare shared or unique virulence factor gene clusters distinguishing persistent and transient strains of S. uberis. A total of 139 S. uberis strains were isolated from three small-holder dairy herds with a high prevalence of S. uberis mastitis. The duration of IMI was used to categorize bacteria into two groups: transient and persistent strains with an IMI duration of less than 1 month and longer than 2 months, respectively. Six representative S. uberis strains, three from each group (transience and persistence) were selected for analysis. All transient strains exhibited multi-locus sequence types (MLST), indicating a highly diverse population of transient S. uberis. In contrast, MLST of persistent strains was available in an online database (pubMLST). Identification of virulence genes was performed using whole-genome sequencing (WGS) data. Differences in genomic size and number of virulent genes were found. For example, the BCA gene or alpha-c protein and the gene associated with capsule formation (hasAB), found in persistent strains, are important for attachment and invasion, as well as the evasion of the antimicrobial mechanisms and survival persistence, respectively. These findings suggest a genetic-level difference between the two strain types. Consequently, a comprehensive study of 139 S. uberis isolates will be conducted to perform an in-depth genetic assessment through WGS analysis on an Illumina platform.

Keywords: Streptococcus Uberis, mastitis, whole genome sequence, intramammary infection, persistent S. Uberis, transient s. Uberis

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Authors: Khin Thanda Win, Chunying Zhang, Sanghyeob Lee

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Mildew resistance locus o (Mlo), a plant-specific gene family with seven-transmembrane (TM), plays an important role in plant resistance to powdery mildew (PM). PM caused by Podosphaera xanthii is a widespread plant disease and probably represents the major fungal threat for many Cucurbits. The recent Cucurbita maxima genome sequence data provides an opportunity to identify and characterize the MLO gene family in this species. Total twenty genes (designated CmaMLO1 through CmaMLO20) have been identified by using an in silico cloning method with the MLO gene sequences of Cucumis sativus, Cucumis melo, Citrullus lanatus and Cucurbita pepo as probes. These CmaMLOs were evenly distributed on 15 chromosomes of 20 C. maxima chromosomes without any obvious clustering. Multiple sequence alignment showed that the common structural features of MLO gene family, such as TM domains, a calmodulin-binding domain and 30 important amino acid residues for MLO function, were well conserved. Phylogenetic analysis of the CmaMLO genes and other plant species reveals seven different clades (I through VII) and only clade IV is specific to monocots (rice, barley, and wheat). Phylogenetic and structural analyses provided preliminary evidence that five genes belonged to clade V could be the susceptibility genes which may play the importance role in PM resistance. This study is the first comprehensive report on MLO genes in C. maxima to our knowledge. These findings will facilitate the functional analysis of the MLOs related to PM susceptibility and are valuable resources for the development of disease resistance in pumpkin.

Keywords: Mildew resistance locus o (Mlo), powdery mildew, phylogenetic relationship, susceptibility genes

Procedia PDF Downloads 165

Authors: Alsu F. Saifitdinova, Alexey S. Komissarov, Svetlana A. Galkina, Elena I. Koshel, Maria M. Kulak, Stephen J. O'Brien, Elena R. Gaginskaya

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The mystery of sex determination is one of the most ancient and still not solved until the end so far. In many species, sex determination is genetic and often accompanied by the presence of dimorphic sex chromosomes in the karyotype. Genomic sequencing gave the information about the gene content of sex chromosomes which allowed to reveal their origin from ordinary autosomes and to trace their evolutionary history. Female-specific W chromosome in birds as well as mammalian male-specific Y chromosome is characterized by the degeneration of gene content and the accumulation of repetitive DNA. Tandem repeats complicate the analysis of genomic data. Despite the best efforts chicken W chromosome assembly includes only 1.2 Mb from expected 55 Mb. Supplementing the information on the sex chromosome composition not only helps to complete the assembly of genomes but also moves us in the direction of understanding of the sex-determination systems evolution. A whole-genome survey to the assembly Gallus_gallus WASHUC 2.60 was applied for repeats search in assembled genome and performed search and assembly of high copy number repeats in unassembled reads of SRR867748 short reads datasets. For cytogenetic analysis conventional methods of fluorescent in situ hybridization was used for previously cloned W specific satellites and specifically designed directly labeled synthetic oligonucleotide DNA probe was used for bioinformatically identified repetitive sequence. Hybridization was performed with mitotic chicken chromosomes and manually isolated giant meiotic lampbrush chromosomes from growing oocytes. A novel chicken W specific satellite (GGAAA)n which is not co-localizes with any previously described classes of W specific repeats was identified and mapped with high resolution. In the composition of autosomes this repeat units was found as a part of upstream regions of gonad specific protein coding sequences. These findings may contribute to the understanding of the role of tandem repeats in sex specific differentiation regulation in birds and sex chromosome evolution. This work was supported by the postdoctoral fellowships from St. Petersburg State University (#1.50.1623.2013 and #1.50.1043.2014), the grant for Leading Scientific Schools (#3553.2014.4) and the grant from Russian foundation for basic researches (#15-04-05684). The equipment and software of Research Resource Center “Chromas” and Theodosius Dobzhansky Center for Genome Bioinformatics of Saint Petersburg State University were used.

Keywords: birds, lampbrush chromosomes, sex chromosomes, tandem repeats

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Authors: Muhammad Adeel Arshad, Faiz-Ul Hassan, Yanfen Cheng

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According to FAO, enteric methane (CH4) production is about 44% of all greenhouse gas emissions from the livestock sector. Ruminants produce CH4 as a result of fermentation of feed in the rumen especially from roughages which yield more CH4 per unit of biomass ingested as compared to concentrates. Efficient ruminal fermentation is not possible without abating CO2 and CH4. Methane abatement strategies are required to curb the predicted rise in emissions associated with greater ruminant production in future to meet ever increasing animal protein requirements. Ecology of ruminal methanogenesis and avenues for its mitigation can be identified through various genomic and transcriptomic techniques. Programs such as Hungate1000 and the Global Rumen Census have been launched to enhance our understanding about global ruminal microbial communities. Through Hungate1000 project, a comprehensive reference set of rumen microbial genome sequences has been developed from cultivated rumen bacteria and methanogenic archaea along with representative rumen anaerobic fungi and ciliate protozoa cultures. But still many species of rumen microbes are underrepresented especially uncultivable microbes. Lack of sequence information specific to the rumen's microbial community has inhibited efforts to use genomic data to identify specific set of species and their target genes involved in methanogenesis. Metagenomic and metatranscriptomic study of entire microbial rumen populations offer new perspectives to understand interaction of methanogens with other rumen microbes and their potential association with total gas and methane production. Deep understanding of methanogenic pathway will help to devise potentially effective strategies to abate methane production while increasing feed efficiency in ruminants.

Keywords: Genome sequences, Hungate1000, methanogens, ruminal fermentation

Procedia PDF Downloads 118

Authors: Faisal Nouroz, Mukaramin Mukaramin

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Of the transposable elements (TEs), the retrotransposons are the most copious elements identified from many sequenced genomes. They have played a major role in genome evolution, rearrangement, and expansions based on their copy and paste mode of proliferation. They are further divided into LTR and Non-LTR retrotransposons. The purpose of the current study was to identify the LTR REs in sequenced Phoenix dactylifera genome and to study their structural diversity. A total of 150 P. dactylifera BAC sequences with > 60kb sizes were randomly retrieved from National Center for Biotechnology Information (NCBI) database and screened for the presence of LTR retrotransposons. Seven bacterial artificial chromosomes (BAC) sequences showed full-length LTR Retrotransposons with 4 Copia and 3 Gypsy families having variable copy numbers in respective families. Reverse transcriptase (RT) domain was found as the most conserved domain among Copia and Gypsy superfamilies and was used to deduce evolutionary analysis. The amino acid residues among various RT sequences showed variability in their percentages indicating post divergence evolution. Amino acid Leucine was found in highest proportions followed by Lysine, while Methionine and Tryptophan were in lowest percentages. The phylogenetic analysis based on RT domains confirmed that although having most conserved RT regions, several evolutionary events occurred causing nucleotide polymorphisms and hence clustering of Gypsy and Copia superfamilies into their respective lineages. The study will be helpful in identification and annotation of these elements in other species and genera and their distribution patterns on chromosomes by fluorescent in situ hybridization techniques.

Keywords: transposable elements, Phoenix dactylifera, retrotransposons, phylogenetic analysis

Procedia PDF Downloads 111

Authors: Naoki Yamamoto, Hidenobu Ozaki, Taiichiro Ookawa, Youming Liu, Kazunori Okada, Aiping Zheng

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Rice sheath blight is one of the destructive fungal diseases in rice. We have thought that rice sheath blight resistance is a polygenic trait. Host-pathogen interactions and secondary metabolites such as lignin and phytoalexins are likely to be involved in defense against R. solani. However, to our knowledge, it is still unknown how sheath blight resistance can be enhanced in rice breeding. To seek for an alternative genetic factor that contribute to sheath blight resistance, we mined relevant allelic variations from rice core collections created in Japan. Based on disease lesion length on detached leaf sheath, we selected 30 varieties of the top tail-end and the bottom tail-end, respectively, from the core collections to perform genome-wide association mapping. Re-sequencing reads for these varieties were used for calling single nucleotide polymorphisms among the 60 varieties to create a SNP panel, which contained 1,137,131 homozygous variant sites after filitering. Association mapping highlighted a locus on the long arm of chromosome 11, which is co-localized with three sheath blight QTLs, qShB11-2-TX, qShB11, and qSBR-11-2. Based on the localization of the trait-associated alleles, we identified an ankyryn repeat-containing protein gene (ANK-M) as an uncharacterized candidate factor for rice sheath blight resistance. Allelic distributions for ANK-M in the whole rice population supported the reliability of trait-allele associations. Gene expression characteristics were checked to evaluiate the functionality of ANK-M. Since an ANK-M homolog (OsPIANK1) in rice seems a basal defense regulator against rice blast and bacterial leaf blight, ANK-M may also play a role in the rice immune system.

Keywords: allele mining, GWAS, QTL, rice sheath blight

Procedia PDF Downloads 56

Authors: Sofia Sehli, Zainab El Ouafi, Casey Eddington, Soumaya Jbara, Kasambula Arthur Shem, Islam El Jaddaoui, Ayorinde Afolayan, Olaitan I. Awe, Allissa Dillman, Hassan Ghazal

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The ability to promptly sequence whole genomes at a relatively low cost has revolutionized the way we study the microbiome. Microbiologists are no longer limited to studying what can be grown in a laboratory and instead are given the opportunity to rapidly identify the makeup of microbial communities in a wide variety of environments. Analyzing whole genome sequencing (WGS) data is a complex process that involves multiple moving parts and might be rather unintuitive for scientists that don’t typically work with this type of data. Thus, to help lower the barrier for less-computationally inclined individuals, TAXAPRO was developed at the first Omics Codeathon held virtually by the African Society for Bioinformatics and Computational Biology (ASBCB) in June 2021. TAXAPRO is an advanced metagenomics pipeline that accurately assembles organelle genomes from whole-genome sequencing data. TAXAPRO seamlessly combines WGS analysis tools to create a pipeline that automatically processes raw WGS data and presents organism abundance information in both a tabular and graphical format. TAXAPRO was evaluated using COVID-19 patient gut microbiome data. Analysis performed by TAXAPRO demonstrated a high abundance of Clostridia and Bacteroidia genera and a low abundance of Proteobacteria genera relative to others in the gut microbiome of patients hospitalized with COVID-19, consistent with the original findings derived using a different analysis methodology. This provides crucial evidence that the TAXAPRO workflow dispenses reliable organism abundance information overnight without the hassle of performing the analysis manually.

Keywords: metagenomics, shotgun metagenomic sequence analysis, COVID-19, pipeline, bioinformatics

Procedia PDF Downloads 184

Authors: Ahmed M. Debela, Mayreli Ortiz , Ciara K. O´Sullivan

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The completion of the human genome Project (HGP) has paved the way for mapping the diversity in the overall genome sequence which helps to understand the genetic causes of inherited diseases and susceptibility to drugs or environmental toxins. Arrayed primer extension (APEX) is a microarray based minisequencing strategy for screening disease causing mutations. It is derived from Sanger DNA sequencing and uses fluorescently dideoxynucleotides (ddNTPs) for termination of a growing DNA strand from a primer with its 3´- end designed immediately upstream of a site where single nucleotide polymorphism (SNP) occurs. The use of DNA polymerase offers a very high accuracy and specificity to APEX which in turn happens to be a method of choice for multiplex SNP detection. Coupling the high specificity of this method with the high sensitivity, low cost and compatibility for miniaturization of electrochemical techniques would offer an excellent platform for detection of mutation as well as sequencing of DNA templates. We are developing an electrochemical APEX for the analysis of SNPs found in the MYH7 gene for group of cardiomyopathy patients. ddNTPs were labeled with four different redox active compounds with four distinct potentials. Thiolated oligonucleotide probes were immobilised on gold and glassy carbon substrates which are followed by hybridisation with complementary target DNA just adjacent to the base to be extended by polymerase. Electrochemical interrogation was performed after the incorporation of the redox labelled dedioxynucleotide. The work involved the synthesis and characterisation of the redox labelled ddNTPs, optimisation and characterisation of surface functionalisation strategies and the nucleotide incorporation assays.

Keywords: array based primer extension, labelled ddNTPs, electrochemical, mutations

Procedia PDF Downloads 224

Authors: Chamilani Nikapitiya, Mahanama De Zoysa, Jehee Lee

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Most of the bacterial diseases are associated with high mortalities in aquaculture species and causing huge economic losses. Different approaches have been taken to prevent or control of bacterial diseases including use of vaccines, probiotics, chemotherapy, water quality management, etc. Antibiotics are widely applying as chemotherapy to control bacterial diseases, however, it has been shown that frequent use of antibiotics is favored to develop multi-drug resistance bacteria. Therefore, phages and phage encoded lytic proteins are known to be one of the most promising alternatives for antibiotics to avoid the emergence of antibiotic-resistant bacteria. We isolated and characterized the two lytic phages, namely pAh-1 and pAs-1 against pathogenic Aeromonas hydrophila and Aeromonas salmonicida, respectively. Morphological characteristics were analyzed by Transmission electron microscopy (TEM) and host strain specificities were tested with Aeromonas and other closely related bacterial strains. TEM analysis revealed that both pAh-1 and pAsm-1 are composed of an icosahedral head and a segmented tail, and we suggest that, they are new members of Myoviridae family. Genome sizes of isolated phages were estimated by restriction enzyme digestion of genomic DNA using selected endonucleases followed by agarose gel electrophoresis. Estimated genome size of pAh-1 and pAs-1 were approximately 64 Kbp and 120 Kbp, respectively. Both pAh-1 and pAs-1 have shown narrow host specificity. Moreover, protective effects of phage therapy against fish pathogenic A. hydrophila were investigated in zebrafish model. The survival rate was 40% higher when zebrafish received intra-peritoneal injection (i.p.) of pAh-1 were simultaneously challenge A. hydrophila (2 x 106 CFU/fish) compared to that without phage treatment. Overall results suggest that both pAh-1 and pAs-1 can be used as a potential phage therapy to control Aeromonas infections in aquaculture.

Keywords: Aeromonas infections, antibiotic resistance, bacteriophage, bio-control, lytic phage

Procedia PDF Downloads 176

Authors: Janos Juhasz, Sandor Pongor, Balazs Ligeti

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Next-generation sequencing, especially whole genome shotgun sequencing, is becoming a common approach to gain insight into the microbiomes in a culture-independent way, even in clinical practice. It does not only give us information about the species composition of an environmental sample but opens the possibility to detect antimicrobial resistance and novel, or currently unknown, pathogens. Accurately and reliably detecting the microbial strains is a challenging task. Here we present a sensitive approach for detecting pathogens in metagenomics samples with special regard to detecting novel variants of known pathogens. We have developed a pipeline that uses fast, short read aligner programs (i.e., Bowtie2/BWA) and comprehensive nucleotide databases. Taxonomic binning is based on the lowest common ancestor (LCA) principle; each read is assigned to a taxon, covering the most significantly hit taxa. This approach helps in balancing between sensitivity and running time. The program was tested both on experimental and synthetic data. The results implicate that our method performs as good as the state-of-the-art BLAST-based ones, furthermore, in some cases, it even proves to be better, while running two orders magnitude faster. It is sensitive and capable of identifying taxa being present only in small abundance. Moreover, it needs two orders of magnitude less reads to complete the identification than MetaPhLan2 does. We analyzed an experimental anthrax dataset (B. anthracis strain BA104). The majority of the reads (96.50%) was classified as Bacillus anthracis, a small portion, 1.2%, was classified as other species from the Bacillus genus. We demonstrate that the evaluation of high-throughput sequencing data is feasible in a reasonable time with good classification accuracy.

Keywords: metagenomics, taxonomy binning, pathogens, microbiome, B. anthracis

Procedia PDF Downloads 113

Authors: Umairah Natasya Binti Mohd Omeershffudin, Suresh Kumar

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Klebsiella pneumoniae, a Gram-negative enteric bacterium that causes nosocomial and urinary tract infections. Particular concern is the global emergence of multidrug-resistant (MDR) strains of Klebsiella pneumoniae. Characterization of antibiotic resistance determinants at the genomic level plays a critical role in understanding, and potentially controlling, the spread of multidrug-resistant (MDR) pathogens. In this study, drug-resistant Klebsiella pneumoniae strain 825795-1 was investigated with extensive computational approaches aimed at identifying novel drug targets among hypothetical proteins. We have analyzed 1099 hypothetical proteins available in genome. We have used in-silico genome subtraction methodology to design potential and pathogen-specific drug targets against Klebsiella pneumoniae. We employed bioinformatics tools to subtract the strain-specific paralogous and host-specific homologous sequences from the bacterial proteome. The sorted 645 proteins were further refined to identify the essential genes in the pathogenic bacterium using the database of essential genes (DEG). We found 135 unique essential proteins in the target proteome that could be utilized as novel targets to design newer drugs. Further, we identified 49 cytoplasmic protein as potential drug targets through sub-cellular localization prediction. Further, we investigated these proteins in the DrugBank databases, and 11 of the unique essential proteins showed druggability according to the FDA approved drug bank databases with diverse broad-spectrum property. The results of this study will facilitate discovery of new drugs against Klebsiella pneumoniae.

Keywords: pneumonia, drug target, hypothetical protein, subtractive genomics

Procedia PDF Downloads 158

Authors: Vyacheslav Furtak, Merja Roivainen, Olga Mirochnichenko, Majid Laassri, Bella Bidzhieva, Tatiana Zagorodnyaya, Vladimir Chizhikov, Konstantin Chumakov

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Molecular epidemiology and environmental surveillance are parts of a rational strategy to control infectious diseases. They have been widely used in the worldwide campaign to eradicate poliomyelitis, which otherwise would be complicated by the inability to rapidly respond to outbreaks and determine sources of the infection. The conventional scheme involves isolation of viruses from patients and the environment, followed by their identification by nucleotide sequences analysis to determine phylogenetic relationships. This is a tedious and time-consuming process that yields definitive results when it may be too late to implement countermeasures. Because of the difficulty of high-throughput full-genome sequencing, most such studies are conducted by sequencing only capsid genes or their parts. Therefore the important information about the contribution of other parts of the genome and inter- and intra-species recombination to viral evolution is not captured. Here we propose a new approach based on the rapid concentration of sewage samples with tangential flow filtration followed by deep sequencing and reconstruction of nucleotide sequences of viruses present in the samples. The entire nucleic acids content of each sample is sequenced, thus preserving in digital format the complete spectrum of viruses. A set of rapid algorithms was developed to separate deep sequence reads into discrete populations corresponding to each virus and assemble them into full-length consensus contigs, as well as to generate a complete profile of sequence heterogeneities in each of them. This provides an effective approach to study molecular epidemiology and evolution of natural viral populations.

Keywords: poliovirus, eradication, environmental surveillance, laboratory diagnosis

Procedia PDF Downloads 254

Authors: Marine Kobalava

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Modern forms of public relations function in an integrated manner together with marketing communication in business companies. This ensures continuity of communication, elimination of duplication in activities, reduction of costs, and strengthening and efficient use of communication means. There exist a number of challenges in implementing integrated forms of public relations in Georgia, especially in terms of marketing communications and strategies. Objectives: The goal of the study is to reveal public relations challenges in Georgian business companies and to develop recommendations along with perfecting marketing communications and strategies. Methodologies: Bibliographic and empirical research has been conducted. Analysis, induction, synthesis, and other methods have been used. Contributions: The challenges of Public relations in Georgia are identified; the perception of different population groups on integrated forms of PR is determined; effective forms of marketing communication are defined; mechanisms for developing marketing strategies are proposed.

Keywords: public relations, challenges, marketing communication, strategy

Procedia PDF Downloads 66

Authors: Andrey V. Khromov, Antonida V. Makhotenko, Ekaterina A. Snigir, Svetlana S. Makarova, Natalia O. Kalinina, Valentin V. Makarov, Mikhail E. Taliansky

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Due to its simplicity, CRISPR/Cas9 has become widely used and capable of inducing mutations in the genes of organisms of various kingdoms. The aim of this work was to develop applications for the efficient modification of DNA coding sequences of phytoene desaturase (PDS), coilin and vacuolar invertase (Solanum tuberosum) genes, and to develop a new nanoparticles carrier efficient technology to deliver the CRISPR/Cas9 system for editing the plant genome. For each of the genes - coilin, PDS and vacuolar invertase, five single RNA guide (sgRNAs) were synthesized. To determine the most suitable nanoplatform, two types of NP platforms were used: magnetic NPs (MNPS) and gold NPs (AuNPs). To test the penetration efficiency, they were functionalized with fluorescent agents - BSA * FITS and GFP, as well as labeled Cy3 small-sized RNA. To measure the efficiency, a fluorescence and confocal microscopy were used. It was shown that the best of these options were AuNP - both in the case of proteins and in the case of RNA. The next step was to check the possibility of delivering components of the CRISPR/Cas9 system to plant cells for editing target genes. AuNPs were functionalized with a ribonucleoprotein complex consisting of Cas9 and corresponding to target genes sgRNAs, and they were biolistically bombarded to axillary buds and apical meristems of potato plants. After the treatment by the best NP carrier, potato meristems were grown to adult plants. DNA isolated from this plants was sent to a preliminary fragment of the analysis to screen out the non-transformed samples, and then to the NGS. The present work was carried out with the financial support from the Russian Science Foundation (grant No. 16-16-04019).

Keywords: biobombardment, coilin, CRISPR/Cas9, nanoparticles, NPs, PDS, sgRNA, vacuolar invertase

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Authors: K. Sathishkumar, V. Thiagarasu

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Due to recent advances in DNA microarray technology, it is now feasible to obtain gene expression profiles of tissue samples at relatively low costs. Many scientists around the world use the advantage of this gene profiling to characterize complex biological circumstances and diseases. Microarray techniques that are used in genome-wide gene expression and genome mutation analysis help scientists and physicians in understanding of the pathophysiological mechanisms, in diagnoses and prognoses, and choosing treatment plans. DNA microarray technology has now made it possible to simultaneously monitor the expression levels of thousands of genes during important biological processes and across collections of related samples. Elucidating the patterns hidden in gene expression data offers a tremendous opportunity for an enhanced understanding of functional genomics. However, the large number of genes and the complexity of biological networks greatly increase the challenges of comprehending and interpreting the resulting mass of data, which often consists of millions of measurements. A first step toward addressing this challenge is the use of clustering techniques, which is essential in the data mining process to reveal natural structures and identify interesting patterns in the underlying data. This work presents an analysis of several clustering algorithms proposed to deals with the gene expression data effectively. The existing clustering algorithms like Support Vector Machine (SVM), K-means algorithm and evolutionary algorithm etc. are analyzed thoroughly to identify the advantages and limitations. The performance evaluation of the existing algorithms is carried out to determine the best approach. In order to improve the classification performance of the best approach in terms of Accuracy, Convergence Behavior and processing time, a hybrid clustering based optimization approach has been proposed.

Keywords: microarray technology, gene expression data, clustering, gene Selection

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Authors: Waqas Shafqat Chattha, Muhammad Iqbal, Amir Shakeel

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Transcriptional factors are proteins that play a vital role in regulating the transcription of target genes in different biological processes and are being widely studied in different plant species. In the current era of genomics, plant genomes sequencing has directed to the genome-wide identification, analyses and categorization of diverse transcription factor families and hence provide key insights into their structural as well as functional diversity. The DNA-binding with One Finger (DOF) proteins belongs to C2-C2-type zinc finger protein family. DOF proteins are plant-specific transcription factors implicated in diverse functions including seed maturation and germination, phytohormone signalling, light-mediated gene regulation, cotton-fiber elongation and responses of the plant to biotic as well as abiotic stresses. In this context, a genome-wide in-silico analysis of DOF TF family in diploid cotton species i.e. Gossypium raimondii has enabled us to identify 55 non-redundant genes encoding DOF proteins renamed as GrDofs (Gossypium raimondii Dof). Gene distribution studies have shown that all of the GrDof genes are unevenly distributed across 12 out of 13 G. raimondii chromosomes. The gene structure analysis illustrated that 34 out of 55 GrDof genes are intron-less while remaining 21 genes have a single intron. Protein sequence-based phylogenetic analysis of putative 55 GrDOFs has divided these proteins into 5 major groups with various paralogous gene pairs. Molecular evolutionary studies aided with the conserved domain as well as gene structure analysis suggested that segmental duplications were the principal contributors for the expansion of Dof genes in G. raimondii.

Keywords: diploid cotton , G. raimondii, phylogenetic analysis, transcription factor

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Authors: Leonard Lipovich, Pattaraporn Thepsuwan, Anton-Scott Goustin, Juan Cai, Donghong Ju, James B. Brown

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Two-thirds of human genes do not encode any known proteins. Aside from long non-coding RNA (lncRNA) genes with recently-discovered functions, the ~40,000 non-protein-coding human genes remain poorly understood, and a role for their transcripts as de-facto unconventional messenger RNAs has not been formally excluded. Ribosome profiling (Riboseq) predicts translational potential, but without independent evidence of proteins from lncRNA open reading frames (ORFs), ribosome binding of lncRNAs does not prove translation. Previously, we mass-spectrometrically documented translation of specific lncRNAs in human K562 and GM12878 cells. We now examined lncRNA translation in human MCF7 cells, integrating strand-specific Illumina RNAseq, Riboseq, and deep mass spectrometry in biological quadruplicates performed at two core facilities (BGI, China; City of Hope, USA). We excluded known-protein matches. UCSC Genome Browser-assisted manual annotation of imperfect (tryptic-digest-peptides)-to-(lncRNA-three-frame-translations) alignments revealed three peptides hypothetically explicable by 'stop-to-nonstop' in-frame replacement of stop codons by amino acids in two ORFs of the lncRNA MMP24-AS1. To search for this phenomenon genomewide, we designed and implemented a novel pipeline, matching tryptic-digest spectra to wildcard-instead-of-stop versions of repeat-masked, six-frame, whole-genome translations. Along with singleton putative stop-to-nonstop events affecting four other lncRNAs, we identified 24 additional peptides with stop-to-nonstop in-frame substitutions from multiple positive-strand MMP24-AS1 ORFs. Only UAG and UGA, never UAA, stop codons were impacted. All MMP24-AS1-matching spectra met the same significance thresholds as high-confidence known-protein signatures. Targeted resequencing of MMP24-AS1 genomic DNA and cDNA from the same samples did not reveal any mutations, polymorphisms, or sequencing-detectable RNA editing. This unprecedented apparent gene-specific violation of the genetic code highlights the importance of matching peptides to whole-genome, not known-genes-only, ORFs in mass-spectrometry workflows, and suggests a new mechanism enhancing the combinatorial complexity of the proteome. Funding: NIH Director’s New Innovator Award 1DP2-CA196375 to LL.

Keywords: genetic code, lncRNA, long non-coding RNA, mass spectrometry, proteogenomics, ribo-seq, ribosome, RNAseq

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Authors: Salman Naveed, Nitant Gandhi, Grant Billings, Zachary Jones, B. Todd Campbell, Michael Jones, Sachin Rustgi

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Cotton (Gossypium spp.) is the primary source of natural fiber in the U.S. and a major crop in the Southeastern U.S. Despite constant efforts to increase the cotton fiber yield, the yield gain has stagnated. Therefore, we undertook a novel approach to improve the cotton fiber yield by altering its growth habit from perennial to annual. In this effort, we identified genotypes with high-expression alleles of five floral induction and meristem identity genes (FT, SOC1, FUL, LFY, and AP1) from an upland cotton mini-core collection and crossed them in various combinations to develop cotton lines with annual growth habit, optimal flowering time and enhanced productivity. To facilitate the characterization of genotypes with the desired combinations of stacked alleles, we identified markers associated with the gene expression traits via genome-wide association analysis using a 63K SNP Array (Hulse-Kemp et al. 2015 G3 5:1187). Over 14,500 SNPs showed polymorphism and were used for association analysis. A total of 396 markers showed association with expression traits. Out of these 396 markers, 159 mapped to genes, 50 to untranslated regions, and 187 to random genomic regions. Biased genomic distribution of associated markers was observed where more trait-associated markers mapped to the cotton D sub-genome. Many quantitative trait loci coincided at specific genomic regions. This observation has implications as these traits could be bred together. The analysis also allowed the identification of candidate regulators of the expression patterns of these floral induction and meristem identity genes whose functions will be validated via virus-induced gene silencing.

Keywords: cotton, GWAS, QTL, expression traits

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Authors: Sandra Smieszek, Jonathan Haines

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Introduction: Numerous research efforts have focused on searching for ‘longevity genes’. However, attempting to decipher the genetic component of the longevous phenotype have resulted in limited success and the mechanisms governing longevity remain to be explained. We conducted a genome-wide homozygosity analysis (GWHA) of the founder population of the Amish community in central Ohio. While genome-wide association studies using unrelated individuals have revealed many interesting longevity associated variants, these variants are typically of small effect and cannot explain the observed patterns of heritability for this complex trait. The Amish provide a large cohort of extended kinships allowing for in depth analysis via family-based approach excellent population due to its. Heritability of longevity increases with age with significant genetic contribution being seen in individuals living beyond 60 years of age. In our present analysis we show that the heritability of longevity is estimated to be increasing with age particularly on the paternal side. Methods: The present analysis integrated both phenotypic and genotypic data and led to the discovery of a series of variants, distinct for stratified populations across ages and distinct for paternal and maternal cohorts. Specifically 5437 subjects were analyzed and a subset of 893 successfully genotyped individuals was used to assess CHIP heritability. We have conducted the homozygosity analysis to examine if homozygosity is associated with increased risk of living beyond 90. We analyzed AMISH cohort genotyped for 614,957 SNPs. Results: We delineated 10 significant regions of homozygosity (ROH) specific for the age group of interest (>90). Of particular interest was ROH on chromosome 13, P < 0.0001. The lead SNPs rs7318486 and rs9645914 point to COL4A2 and our lead SNP. COL25A1 encodes one of the six subunits of type IV collagen, the C-terminal portion of the protein, known as canstatin, is an inhibitor of angiogenesis and tumor growth. COL4A2 mutations have been reported with a broader spectrum of cerebrovascular, renal, ophthalmological, cardiac, and muscular abnormalities. The second region of interest points to IRS2. Furthermore we built a classifier using the obtained SNPs from the significant ROH region with 0.945 AUC giving ability to discriminate between those living beyond to 90 years of age and beyond. Conclusion: In conclusion our results suggest that a history of longevity does indeed contribute to increasing the odds of individual longevity. Preliminary results are consistent with conjecture that heritability of longevity is substantial when we start looking at oldest fifth and smaller percentiles of survival specifically in males. We will validate all the candidate variants in independent cohorts of centenarians, to test whether they are robustly associated with human longevity. The identified regions of interest via ROH analysis could be of profound importance for the understanding of genetic underpinnings of longevity.

Keywords: regions of homozygosity, longevity, SNP, Amish

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Authors: Igor V. Grigoriev

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Understanding of biological processes encoded in fungi is instrumental in addressing future food, feed, and energy demands of the growing human population. Genomics is a powerful and quickly evolving tool to understand these processes. The Fungal Genomics Program of the US Department of Energy Joint Genome Institute (JGI) partners with researchers around the world to explore fungi in several large scale genomics projects, changing the fungal genomics landscape. The key trends of these changes include: (i) rapidly increasing scale of sequencing and analysis, (ii) developing approaches to go beyond culturable fungi and explore fungal ‘dark matter,’ or unculturables, and (iii) functional genomics and multi-omics data integration. Power of comparative genomics has been recently demonstrated in several JGI projects targeting mycorrhizae, plant pathogens, wood decay fungi, and sugar fermenting yeasts. The largest JGI project ‘1000 Fungal Genomes’ aims at exploring the diversity across the Fungal Tree of Life in order to better understand fungal evolution and to build a catalogue of genes, enzymes, and pathways for biotechnological applications. At this point, at least 65% of over 700 known families have one or more reference genomes sequenced, enabling metagenomics studies of microbial communities and their interactions with plants. For many of the remaining families no representative species are available from culture collections. To sequence genomes of unculturable fungi two approaches have been developed: (a) sequencing DNA from fruiting bodies of ‘macro’ and (b) single cell genomics using fungal spores. The latter has been tested using zoospores from the early diverging fungi and resulted in several near-complete genomes from underexplored branches of the Fungal Tree, including the first genomes of Zoopagomycotina. Genome sequence serves as a reference for transcriptomics studies, the first step towards functional genomics. In the JGI fungal mini-ENCODE project transcriptomes of the model fungus Neurospora crassa grown on a spectrum of carbon sources have been collected to build regulatory gene networks. Epigenomics is another tool to understand gene regulation and recently introduced single molecule sequencing platforms not only provide better genome assemblies but can also detect DNA modifications. For example, 6mC methylome was surveyed across many diverse fungi and the highest among Eukaryota levels of 6mC methylation has been reported. Finally, data production at such scale requires data integration to enable efficient data analysis. Over 700 fungal genomes and other -omes have been integrated in JGI MycoCosm portal and equipped with comparative genomics tools to enable researchers addressing a broad spectrum of biological questions and applications for bioenergy and biotechnology.

Keywords: fungal genomics, single cell genomics, DNA methylation, comparative genomics

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Authors: Walid Louhichi, Mouhebeddine Berrima, Narjes Ben Rajed

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Elasticity is the essential property of cloud computing. As the name suggests, it constitutes the ability of a cloud system to adjust resource provisioning in relation to fluctuating workload. There are two types of elasticity operations, vertical and horizontal. In this work, we are interested in horizontal scaling, which is ensured by two mechanisms; scaling in and scaling out. Following the sizing of the system, we can adopt scaling in in the event of over-supply and scaling out in the event of under-supply. In this paper, we propose a formal model, based on colored and temporized Petri nets, for the modeling of the duplication and the removal of a virtual machine from a server. This model is based on formal Petri Nets modeling language. The proposed models are edited, verified, and simulated with two examples implemented in CPNtools, which is a modeling tool for colored and timed Petri nets.

Keywords: cloud computing, elasticity, elasticity controller, petri nets, scaling in, scaling out

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Authors: Kunyaporn Pipithsangchan, Alongkorn Korntong, Assanee Songserm, Phatchara Piriyavinit, Saowanee Dechakampoo

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The Kingdom of Thailand is one of the South East Asian countries. From its area of 514,000 square kilometers (51 million ha), at least 18,000 plant species (8% of the world total) have been estimated to be found in the country. As a result, the conservation of plant genetic diversity, particularly food crops, is becoming important and is an assurance for the national food security. Department of Agriculture Genebank or DOA Genebank, Thailand is responsible for the conservation of plant germplasm by participating and accomplishing several collaborative projects both at national and international levels. Integrated Management System of Plant Genetic Resources or IMPGR is one of the most outstandingly successful cooperation. It is a multilateral project under the Asian Food and Agriculture Cooperation Initiative (AFACI) supported by the Rural Development Administration (RDA) of South Korea. The member countries under the project consist of 11 nations namely Bangladesh, Cambodia, Indonesia, Laos PDR, Mongolia, Nepal, Philippines, Sri Lanka, Thailand, Vietnam and South Korea. The project enabled the members to jointly address the global issues in plant genetic resource (PGR) conservation and strengthen their network in this aspect. The 1st phase of IMPGR project, entitled 'Collection, Conservation, Regeneration and Characterization of Cucurbitaceae and Solanaceae 2012-2014', comprises three main objectives that are: 1) To improve management in storage facilities, collection, and regeneration, 2) To improve linkage between Genebank and material sources (for regeneration), and 3) To improve linkage between Genebank and other field crop or/and horticultural research centers. The project was done for three years from 2012 to 2014. The activities of the project can be described as following details: In the 1st year, there were 9 target provinces for completing plant genetic resource survey and collection. 108 accessions of PGR were collected. In the 2nd year, PGR were continuously surveyed and collected from 9 provinces. The total number of collection was 140 accessions. In addition, the process of regeneration of 237 accessions collected from 1st and 2nd year was started at several sites namely Biotechnology Research and Development Office, Sukothai Horticultural Research Center, Tak Research, and Development Center and Nakhon Ratchasima Research and Development Center. In the 3rd year, besides survey and collection of 115 accessions from 9 target provinces, PGR characterization and evaluation were done for 206 accessions. Moreover, safety duplication of 253 PGR at the World Seed Vault, RDA, was also done according to Standard Agreement on Germplasm Safety Duplication between Department of Agriculture, Ministry of Agriculture and Cooperatives, the Kingdom of Thailand and the National Agrobiodiversity Center, Rural Development Administration of the Republic of Korea. The success of the 1st phase project led to the second phase which entitled 'Collection and Characterization for Effective Conservation of Local Capsicum spp., Solanum spp. and Lycopersicon spp. in Thailand 2015-2017'.

Keywords: characterization, conservation, DOA genebank, plant genetic resources

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Authors: S. Fahiminiya, J. Nadaf, F. Rauch, L. Jerome-Majewska, J. Majewski

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Background: Sequencing of protein coding regions of human genome (Whole Exome Sequencing; WES), has demonstrated a great success in the identification of causal mutations for several rare genetic disorders in human. Generally, most of WES studies have focused on rare variants in coding exons and splicing-sites where missense substitutions lead to the alternation of protein product. Although focusing on this category of variants has revealed the mystery behind many inherited genetic diseases in recent years, a subset of them remained still inconclusive. Here, we present the result of our WES studies where analyzing only rare variants in coding regions was not conclusive but further investigation revealed the involvement of non-coding variants and copy number variations (CNV) in etiology of the diseases. Methods: Whole exome sequencing was performed using our standard protocols at Genome Quebec Innovation Center, Montreal, Canada. All bioinformatics analyses were done using in-house WES pipeline. Results: To date, we successfully identified several disease causing mutations within gene coding regions (e.g. SCARF2: Van den Ende-Gupta syndrome and SNAP29: 22q11.2 deletion syndrome) by using WES. In addition, we showed that variants in non-coding regions and CNV have also important value and should not be ignored and/or filtered out along the way of bioinformatics analysis on WES data. For instance, in patients with osteogenesis imperfecta type V and in patients with glucocorticoid deficiency, we identified variants in 5'UTR, resulting in the production of longer or truncating non-functional proteins. Furthermore, CNVs were identified as the main cause of the diseases in patients with metaphyseal dysplasia with maxillary hypoplasia and brachydactyly and in patients with osteogenesis imperfecta type VII. Conclusions: Our study highlights the importance of considering non-coding variants and CNVs during interpretation of WES data, as they can be the only cause of disease under investigation.

Keywords: whole exome sequencing data, non-coding variants, copy number variations, rare diseases

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Authors: Zewdneh Zana Zate

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The study focuses on the evolutionary and ecological genomics of both wild and cultivated Coffea arabica L. at its center of origin, Ethiopia, aiming to uncover how this vital species may withstand future climate changes. Utilizing bioclimatic models, we project the future distribution of Arabica under varied climate scenarios for 2050 and 2080, identifying potential conservation zones and immediate risk areas. Through whole-genome resequencing of accessions from Ethiopian gene banks, this research assesses genetic diversity and divergence between wild and cultivated populations. It explores relationships, demographic histories, and potential hybridization events among Coffea arabica accessions to better understand the species' origins and its connection to parental species. This genomic analysis also seeks to detect signs of natural or artificial selection across populations. Integrating these genomic discoveries with ecological data, the study evaluates the current and future ecological and genomic vulnerabilities of wild Coffea arabica, emphasizing necessary adaptations for survival. We have identified key genomic regions linked to environmental stress tolerance, which could be crucial for breeding more resilient Arabica varieties. Additionally, our ecological modeling predicted a contraction of suitable habitats, urging immediate conservation actions in identified key areas. This research not only elucidates the evolutionary history and adaptive strategies of Arabica but also informs conservation priorities and breeding strategies to enhance resilience to climate change. By synthesizing genomic and ecological insights, we provide a robust framework for developing effective management strategies aimed at sustaining Coffea arabica, a species of profound global importance, in its native habitat under evolving climatic conditions.

Keywords: coffea arabica, climate change adaptation, conservation strategies, genomic resilience

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Authors: Pavlina Capkova, Josef Srovnal, Vera Becvarova, Marie Trkova, Zuzana Capkova, Andrea Stefekova, Vaclava Curtisova, Alena Santava, Sarka Vejvalkova, Katerina Adamova, Radek Vodicka

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ASDs are heterogeneous and complex developmental diseases with a significant genetic background. Recurrent CNVs are known to be a frequent cause of ASD. These CNVs can have, however, a variable expressivity which results in a spectrum of phenotypes from asymptomatic to ID/DD/ASD. ASD is associated with ID in ~75% individuals. Various platforms are used to detect pathogenic mutations in the genome of these patients. The performed study is focused on a determination of the frequency of pathogenic mutations in a group of ASD patients and a group of ID/DD patients using various strategies along with a comparison of their detection rate. The possible role of the origin of these mutations in aetiology of ASD was assessed. The study included 35 individuals with ASD and 68 individuals with ID/DD (64 males and 39 females in total), who underwent rigorous genetic, neurological and psychological examinations. Screening for pathogenic mutations involved karyotyping, screening for FMR1 mutations and for metabolic disorders, a targeted MLPA test with probe mixes Telomeres 3 and 5, Microdeletion 1 and 2, Autism 1, MRX and a chromosomal microarray analysis (CMA) (Illumina or Affymetrix). Chromosomal aberrations were revealed in 7 (1 in the ASD group) individuals by karyotyping. FMR1 mutations were discovered in 3 (1 in the ASD group) individuals. The detection rate of pathogenic mutations in ASD patients with a normal karyotype was 15.15% by MLPA and CMA. The frequencies of the pathogenic mutations were 25.0% by MLPA and 35.0% by CMA in ID/DD patients with a normal karyotype. CNVs inherited from asymptomatic parents were more abundant than de novo changes in ASD patients (11.43% vs. 5.71%) in contrast to the ID/DD group where de novo mutations prevailed over inherited ones (26.47% vs. 16.18%). ASD patients shared more frequently their mutations with their fathers than patients from ID/DD group (8.57% vs. 1.47%). Maternally inherited mutations predominated in the ID/DD group in comparison with the ASD group (14.7% vs. 2.86 %). CNVs of an unknown significance were found in 10 patients by CMA and in 3 patients by MLPA. Although the detection rate is the highest when using CMA, recurrent CNVs can be easily detected by MLPA. CMA proved to be more efficient in the ID/DD group where a larger spectrum of rare pathogenic CNVs was revealed. This study determined that maternally inherited highly penetrant mutations and de novo mutations more often resulted in ID/DD without ASD in patients. The paternally inherited mutations could be, however, a source of the greater variability in the genome of the ASD patients and contribute to the polygenic character of the inheritance of ASD. As the number of the subjects in the group is limited, a larger cohort is needed to confirm this conclusion. Inherited CNVs have a role in aetiology of ASD possibly in combination with additional genetic factors - the mutations elsewhere in the genome. The identification of these interactions constitutes a challenge for the future. Supported by MH CZ – DRO (FNOl, 00098892), IGA UP LF_2016_010, TACR TE02000058 and NPU LO1304.

Keywords: autistic spectrum disorders, copy number variant, chromosomal microarray, intellectual disability, karyotyping, MLPA, multiplex ligation-dependent probe amplification

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Authors: Fahimeh Palizban, Farshad Noravesh, Amir Hossein Saeidian, Mahya Mehrmohamadi

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In the recent decade, the emergence of liquid biopsy has significantly improved cancer monitoring and detection. Dying cells, including those originating from tumors, shed their DNA into the blood and contribute to a pool of circulating fragments called cell-free DNA. Accordingly, identifying the tissue origin of these DNA fragments from the plasma can result in more accurate and fast disease diagnosis and precise treatment protocols. Open chromatin regions are important epigenetic features of DNA that reflect cell types of origin. Profiling these features by DNase-seq, ATAC-seq, and histone ChIP-seq provides insights into tissue-specific and disease-specific regulatory mechanisms. There have been several studies in the area of cancer liquid biopsy that integrate distinct genomic and epigenomic features for early cancer detection along with tissue of origin detection. However, multimodal analysis requires several types of experiments to cover the genomic and epigenomic aspects of a single sample, which will lead to a huge amount of cost and time. To overcome these limitations, the idea of predicting OCRs from WGS is of particular importance. In this regard, we proposed a computational approach to target the prediction of open chromatin regions as an important epigenetic feature from cell-free DNA whole genome sequence data. To fulfill this objective, local sequencing depth will be fed to our proposed algorithm and the prediction of the most probable open chromatin regions from whole genome sequencing data can be carried out. Our method integrates the signal processing method with sequencing depth data and includes count normalization, Discrete Fourie Transform conversion, graph construction, graph cut optimization by linear programming, and clustering. To validate the proposed method, we compared the output of the clustering (open chromatin region+, open chromatin region-) with previously validated open chromatin regions related to human blood samples of the ATAC-DB database. The percentage of overlap between predicted open chromatin regions and the experimentally validated regions obtained by ATAC-seq in ATAC-DB is greater than 67%, which indicates meaningful prediction. As it is evident, OCRs are mostly located in the transcription start sites (TSS) of the genes. In this regard, we compared the concordance between the predicted OCRs and the human genes TSS regions obtained from refTSS and it showed proper accordance around 52.04% and ~78% with all and the housekeeping genes, respectively. Accurately detecting open chromatin regions from plasma cell-free DNA-seq data is a very challenging computational problem due to the existence of several confounding factors, such as technical and biological variations. Although this approach is in its infancy, there has already been an attempt to apply it, which leads to a tool named OCRDetector with some restrictions like the need for highly depth cfDNA WGS data, prior information about OCRs distribution, and considering multiple features. However, we implemented a graph signal clustering based on a single depth feature in an unsupervised learning manner that resulted in faster performance and decent accuracy. Overall, we tried to investigate the epigenomic pattern of a cell-free DNA sample from a new computational perspective that can be used along with other tools to investigate genetic and epigenetic aspects of a single whole genome sequencing data for efficient liquid biopsy-related analysis.

Keywords: open chromatin regions, cancer, cell-free DNA, epigenomics, graph signal processing, correlation clustering

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Authors: Kaddi Mohammed, A. Benatiallah, D. Benatiallah

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Data grid is a technology that has full emergence of new challenges, such as the heterogeneity and availability of various resources and geographically distributed, fast data access, minimizing latency and fault tolerance. Researchers interested in this technology address the problems of the various systems related to the industry such as task scheduling, load balancing and replication. The latter is an effective solution to achieve good performance in terms of data access and grid resources and better availability of data cost. In a system with duplication, a coherence protocol is used to impose some degree of synchronization between the various copies and impose some order on updates. In this project, we present an approach for placing replicas to minimize the cost of response of requests to read or write, and we implement our model in a simulation environment. The placement techniques are based on a cost model which depends on several factors, such as bandwidth, data size and storage nodes.

Keywords: response time, query, consistency, bandwidth, storage capacity, CERN

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Authors: Aishwarya Shekhar, Himanshu Sharma

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Data deduplication is one of important data compression techniques for eliminating duplicate copies of repeating data, and has been widely used in cloud storage to reduce the amount of storage space and save bandwidth. In this process, duplicate data is expunged, leaving only one copy means single instance of the data to be accumulated. Though, indexing of each and every data is still maintained. Data deduplication is an approach for minimizing the part of storage space an organization required to retain its data. In most of the company, the storage systems carry identical copies of numerous pieces of data. Deduplication terminates these additional copies by saving just one copy of the data and exchanging the other copies with pointers that assist back to the primary copy. To ignore this duplication of the data and to preserve the confidentiality in the cloud here we are applying the concept of hybrid nature of cloud. A hybrid cloud is a fusion of minimally one public and private cloud. As a proof of concept, we implement a java code which provides security as well as removes all types of duplicated data from the cloud.

Keywords: confidentiality, deduplication, data compression, hybridity of cloud

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Authors: Nemat Sokhandan Bashir

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Threats from viruses to agricultural crops could be even larger than the losses caused by the other pathogens because, in many cases, the viral infection is latent but crucial from an epidemic point of view. Wild vegetation can be a source of many viruses that eventually find their destiny in crop plants. Although often asymptomatic in wild plants due to adaptation, they can potentially cause serious losses in crops. Therefore, exploring viruses in wild vegetation is very important. Recently, omics have been quite useful for exploring plant viruses from various plant sources, especially wild vegetation. For instance, we have discovered viruses such as Ambrossia asymptomatic virus I (AAV-1) through the application of metagenomics from Oklahoma Prairie Reserve. Accordingly, extracts from randomly-sampled plants are subjected to high speed and ultracentrifugation to separated virus-like particles (VLP), then nucleic acids in the form of DNA or RNA are extracted from such VLPs by treatment with phenol—chloroform and subsequent precipitation by ethanol. The nucleic acid preparations are separately treated with RNAse or DNAse in order to determine the genome component of VLPs. In the case of RNAs, the complementary cDNAs are synthesized before submitting to DNA sequencing. However, for VLPs with DNA contents, the procedure would be relatively straightforward without making cDNA. Because the length of the nucleic acid content of VPLs can be different, various strategies are employed to achieve sequencing. Techniques similar to so-called "chromosome walking" may be used to achieve sequences of long segments. When the nucleotide sequence data were obtained, they were subjected to BLAST analysis to determine the most related previously reported virus sequences. In one case, we determined that the novel virus was AAV-l because the sequence comparison and analysis revealed that the reads were the closest to the Indian citrus ringspot virus (ICRSV). AAV—l had an RNA genome with 7408 nucleotides in length and contained six open reading frames (ORFs). Based on phylogenies inferred from the replicase and coat protein ORFs of the virus, it was placed in the genus Mandarivirus.

Keywords: wild, plant, novel, metagenomics

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Authors: Tomokazu Shirai, Akihiko Kondo

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Technologies of metabolic engineering or synthetic biology are essential for effective microbial bioproduction. It is especially important to develop an in silico tool for designing a metabolic pathway producing an unnatural and valuable chemical such as fossil materials of fuel or plastics. We here demonstrated two in silico tools for designing novel metabolic pathways: BioProV and HyMeP. Furthermore, we succeeded in creating an artificial metabolic pathway by enzyme engineering.

Keywords: bioinformatics, metabolic engineering, synthetic biology, genome scale model

Procedia PDF Downloads 318