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Volume 2 Number 1 2008 
CONTENTS AND ABSTRACTS
Kazuharu Arakawa, Haruo Suzuki, Masaru Tomita (Japan) Computational Genome Analysis Using The G-language System (pp 1-13)
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Invited Review: Computational genome analysis requires sophisticated workflows, seamlessly uniting multiple tools and algorithms. In order to maximize the productivity of genomics research with bioinformatics, a computational framework that allows rapid integration of available resources is desirable. G-language Genome Analysis Environment is a generic open-source workbench for this purpose, with the aim to: 1) construct an integrated analysis and development environment for bioinformatics, 2) systematically accumulate and implement existing algorithms and data, and to 3) aid the construction of analysis workflows. This system provides over 200 analysis methods for genome informatics and systems biology, with programmable interfaces, an interactive command-line shell, and a graphical user interface. Here we review the methods and algorithms implemented in this system especially focusing on genome informatics analysis, including methods for the identification of sequences with significant information content using information theory, observation of nucleotide composition and genomic compositional asymmetry, calculation of codon bias measures and prediction of gene expression levels, and statistical analysis of short oligomers such as short tandem repeats and palindromes. Since these methods are combined with several other applications and algorithms to produce a workflow in genome informatics research for studying specific biological questions, we also present brief overviews of workflows utilizing these algorithms used in several genomic studies.
Hüseyin Avni Öktem, Füsun Eyidoğan, Feyza Selçuk, Mehmet Tufan Öz (Turkey), Jaime A. Teixeira da Silva (Japan), Meral Yücel (Turkey) Revealing Response of Plants to Biotic and Abiotic Stresses with Microarray Technology (pp 14-48)
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Invited Review: DNA microarrays became a widely employed tool in functional genomics and global gene expression analysis. They have been intensely used to investigate plant transcriptomes to answer various biological questions involving stress response and tolerance. Identification of stress-related genes via microarrays provides valuable information to improve biotic and abiotic stress tolerance in plants. Since these stresses are the major factors that limit plant growth and production worldwide, in this review recent progresses resulting from gene expression analysis using microarrays under biotic and abiotic stresses are summarized. Moreover microarray technology, manufacturing of arrays, experimental approaches in cDNA and oligonucleotide microarray platforms and data analysis, which are followed by microarray expression profiling studies in plant sciences, are briefly explained.
S. D. Puthucheary, Chai Hwa Chia, Chua Kek Heng (Malaysia) Investigation of Differential Gene Expression Patterns of Virulent and Avirulent Burkholderia Species (pp 49-52)
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Original Research Paper: A differential gene expression study revealed that Burkholderia pseudomallei, a virulent species, and B. thailandensis, an avirulent species, had similar gene expression patterns compared to another virulent species, B. cepacia. Only the differentially expressed gene fragments from B. cepacia were sequenced and the four fragments were amplified by arbitrary primers ACP5, ACP9, ACP10 and ACP12. The genes were identified as expressing lytic transglycosylase, porin, outer membrane autotransporter barrel domain, and two component transcriptional regulator of the LuxR family, with a 95, 94, 100, and 90% identity respectively, against B. cenocepacia HI2424 and AU 1054 nucleotides. Although the genes encoding these proteins are shared by most bacteria, certain factors such as growth conditions might affect the expression of the genes in some bacteria, as down regulation of gene expression in B. pseudomallei and B. thailandensis was noticed in this study.
Mohamed Ibrahim Motawei (Egypt) Selection of Wheat Quality Genotypes Using Molecular Markers for High Molecular Weight Glutenin Alleles at the Glu-B1 Locus (pp 53-56)
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Original Research Paper: Wheat cultivar ‘Yeocra Rojo’ and ten selected genotypes originating from immature embryo culture of this cultivar were evaluated for their productivity in two field experiments during 2005/2006 and 2006/2007 winter seasons. Wheat genotypes YR-9 and YR-10 exhibited the tallest plants whereas the shortest plants were those of cv. ‘Yeocra Rojo’. In general, genotypes YR-2, YR-7, and YR-10 produced the highest grain yield while YR-8 produced the lowest. YR-8 also gave the lowest harvest index. YR-7 had the most grains per spike, spike length and number of spikelets per spike compared to its parent cultivar and all other genotypes. A set of PCR-based markers for specific HMW glutenin genes encoding By-subunits were used to identify wheat genotypes carrying By genes at the Glu-B1 locus for its bread-making quality. The presence of the gene encoding By8, which exists in the allele combination Glu-B1b (Bx7 + By8), was detected only in one genotype: YR-7. Primer pair ZSBy9aF1/R3 gave characteristic banding patterns for Glu-B1c (Bx7+By9) and can therefore be used to discriminate By9-containing alleles from non-By9 alleles. Primer pair ZSBy9F2/R2 produced individuals with a diagnostic banding pattern for allele Glu-B1f (Bx13+By16) in genotype YR-10 while YR-8 and YR-9 did not produce any PCR product that was found to be specific for By-null or the 20 gene. YR-7 and YR-10 produced the highest grain yield and had the By8 and By16 genes, respectively, which are associated with superior bread-making quality. YR-9, however, produced high grain yield but had the By-null or 20 gene, which is associated with poor bread-making quality. Therefore, fast and accurate identification of By genes by molecular markers at the Glu-B1 locus could be an efficient way for early selection of useful wheat genotypes with good bread-making quality.
Ajay Kumar Mishra, Kamal Sharma, Raj Shekhar Misra (India) Rapid and Efficient Method for the Extraction of Fungal and Oomycetes Genomic DNA (pp 57-59)
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Techniques Paper: An improved protocol for the isolation of high-quality DNA from fungi is described. The method involves inactivating proteins by SDS/proteinase K and precipitating polysaccharides in the presence of high salt. Further purification is based on differential solubility of DNA and high molecular weight polysaccharides in aqueous media. The procedure does not use the toxic and potentially hazardous chemical such as phenol and as many as 50 samples can be processed per day. The purity of isolated genomic DNA was confirmed by means of spectrophotometer analysis (A260/280 ratio of 1.80-1.96), indicated a minimal presence of contaminating metabolites. The method yielded 0.55-0.92 µg DNA mg-1 freeze dried mycelia, when tested on three fungal species Fusarium solani, Colletotrichum capsici, and Rhizoctonia solani and two oomycetes Phytophthora colocasiae and Pythium aphanidermatum. The DNA was completely digested with EcoRI and HindIII. PCR-based technique such as random amplification of polymorphic DNA (RAPD), showed the DNA’s compatibility with downstream applications.
Muthulekshmi Lajapathy Jeeva, Kamal Sharma, Ajay Kumar Mishra, Raj Shekhar Misra (India) Rapid Extraction of Genomic DNA from Sclerotium rolfsii Causing Collar Rot of Amorphophallus (pp 60-62)
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Techniques paper: Current methods for the extraction of genomic DNA from Sclerotium rolfsii, a Basidiomycetous fungal pathogen, are often time-consuming and yield poor quantity and quality of DNA. The high mucilage and polysaccharide content in this fungus add difficulties in genomic DNA isolation, and further down stream applications. We therefore investigated a new and rapid DNA isolation method, which involves inactivation of contaminant proteins by using CTAB/Proteinase K and precipitation of polysaccharides and proteins in the presence of a high concentration of salt. This protocol yielded 0.89 ± 0.10 µg DNA mg-1 of mycelium from S. rolfsii with purity ranges from 1.83-1.95 as confirmed by A260/280 and A260/230 spectrophotometric readings. The new protocol can be successfully used for both small and large-scale preparation of genomic DNA which is highly suitable for further downstream processes like PCR-RAPD and ITS amplification of the rDNA-ITS region.
Kamel A. Abd-Elsalam, Ali H. Bahkali, Abdulaziz A. Al-Khedhairy (Saudi Arabia), Joseph-Alexander Verreet (Germany) Development of a Conventional and Lightcycler PCR assay for Detection of Fusarium solani (pp 63-67)
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Original Research Paper: Early, precise detection and identification of soil-borne fungi are essential for efficient plant disease management. Three sets of specific primers were designed from the internal transcribed spacer (ITS) regions, ITS-Fs-forward and ITS-Fs-reverse, of the rRNA gene to identify Fusarium solani isolates in pure mycelial culture. The specificity and suitability of the PCR techniques has been tested with different cotton fungal pathogens, including various Fusarium species and 29 F. solani isolates. ITS-Fs7 and ITS-FS8 primers amplified two fragments (480 and 390 bp) from F. solani genomic DNA, and the ITS-Fs2 primer set amplified two different sizes (540 and 420 bp) of fragments. The 390 bp- and 420 bp- fragments were specific to F. solani. The primer sets amplified none of fragments from Rhizoctonia solani and Macrophomina phaseolina. In the assay, SYBR Green I was used as fluorescent dye enabling real-time detection of PCR products. Characterization of the amplicons was achieved by melting point analysis (85 ± 0.1°C). In conventional PCR the detection limit of all primers was 100 pg, whereas detection threshold of the LightCycler PCR were 100 fg of genomic DNA from F. solani mycelium. Real-time PCR assay allows a more reliable and rapid detection of F. solani which is considerably faster than conventional detection assays. The present PCR test represents a rapid and reliable method for genetically based identification of F. solani cultures. To the best of our knowledge, this is the first report describing the application of real-time PCR assay for the identification of F. solani isolates recovered from cotton.
Rajinder S. Ranu, Shyamal Chakrabarti, Lysa-Anne M. Volpe, Bridget Brown, Dong Wang, Jianguo Fan (USA) 1-Aminocyclopropane-1-carboxylate (ACC) Synthases of Rosa hybrida: Analysis of Genomic Gene Structure and the Cis-Acting Regulatory Elements in their Promoters (pp 68-76)
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Original Research Paper: The phytohormone ethylene is involved in the modulation of a variety of growth and developmental processes in plants, including fruit ripening. Many forms of visual changes observed in rose flowers, including flower opening, petal senescence and changes in floral scent emission are correlated to ethylene levels in flowers. As 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) is one of the key regulatory enzymes in ethylene biosynthesis, ACS genes have been intensively investigated. Here we describe the structure of three full-length ACS genomic clones from Rosa hybrida cv. ‘Kardinal’. These genes contain four exons and three introns and share sequence homologies with other plant ACSs with typical features that are characteristic of all ACSs. Plants selectively activate genes via interaction between transcription factor(s) and their specific binding motifs located in the genes’ promoters. To identify/analyze cis-acting elements/motifs located in promoters of ACS genes, we have taken a computational approach using PLACE and AGRIS databases on the assumption that commonalities of cis-regulatory elements in the promoters are related in each gene to their expression in response to a particular signal. The resulting ethylene related cis-elements have been identified. The relative positions of these common regulatory elements vary among these promoters suggesting that protein-protein interactions among transcription factors may be another factor(s) in determining differential gene regulation. In future, as more full-length ACS genes from the Rosa multi-gene family are identified, a better picture of their differential regulation will emerge. This knowledge may allow the development of new rose cultivars with desirable characteristics through genetic manipulations/modifications.
Ali H. Bahkali, Kamel A. Abd-Elsalam, Mohamed A. Moslem (Saudi Arabia) In-house Protocol for Isolation of Restrictable and Amplifiable Genomic DNA from Plants, Fungi and Bacteria (pp 77-80)
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Original Research Paper: Several rapid DNA isolation protocols are not available for plant and microorganisms with the same method. The method was applied to 5 plant species, 4 fungal species and 4 bacterial species. Optimal extraction was achieved by incorporating an RNAse A and proteinase K enzymatic digestion step. The protocol produced an acceptable quantity of high-quality DNA. Up to 50 µg of DNA were routinely obtained from a minimal amount of 100 mg of fungal bacterial and plant sample. Fungal DNA prepared by this method was used as a template for PCR to amplify the internal transcribed spacer (ITS) and microsatellite-primed PCR and gave reproducible patterns. The amplicon length of the fragment ITS1/ITS4, ranged in size from 620 to 700 bp. The amplified PCR products of ITS regions in plants ranged in size from 550 to 700 bp, indicating polymorphisms of size in this region. The resultant amplicon was then incubated with the restriction endonucleases RsaI and CofI prior to analysis by gel electrophoresis. The protocol presented here offers an interesting and efficient alternative, eliminating most expensive kits, discounting the material cost per sample to $0.10.
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