Construction Of Genomic Library Research Articles

Cloning of triose phosphate isomerase gene from an antarctic psychrophilic Pseudomonas sp. by degenerate and splinkerette PCR

Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterial isolate codenamed π9 was selected for the cloning of the gene encoding triose phosphate isomerase (TIM), an enzyme in the glycolytic pathway. Based on 16S rRNA gene sequence analysis, this isolate was identified as a species of the genus Pseudomonas under the P. fluorescens group. The cloning of a 816bp fragment of TIM gene which covers the 756bp open reading frame was achieved by a combination of degenerate and splinkerette PCRs. The partial sequence of this gene was first PCR amplified by using degenerate primers and the flanking sequences were subsequently amplified by splinkerette PCR technique. Amino acid sequence of the cloned TIM was 97% identical to TIM from Pseudomonas fluorescens and shared 51% identity with the TIM from psychrophilic Vibrio sp. This work demonstrated the use of multiple PCR techniques to clone a gene without prior knowledge of its sequence. The cloning of the TIM gene by PCR was more rapid and cost effective compared to the traditional genomic library construction and screening method. Homology model of the TIM protein in this study was generated based on Escherichia coli TIM crystal structure. The model could serve as a hypothetical TIM structure from a psychrophilic microorganism for further investigation into areas that showed deviations from the known mesophilic TIM structures.

Construction of Genomic DNA Library of Korean Ginseng (Panax ginseng C. A. MEYER) and Development of Sequence-Tagged Sites

This study describes an efficient approach for developing sequence tagged sites (STS) for Panax ginseng C.A. MEYER, and their applications for line discrimination. By using the methylation filtering (MF) technique, a genomic library was constructed, in which clone inserts were derived from the hypomethylated regions of ginseng genome. A methylation unfiltered genomic library was also constructed and the clone inserts were compared to those from the MF library in terms of sequence characteristics. Sequence analysis revealed that MF efficiently enriched the protein coding region of P. ginseng, for which the repetitive DNA appeared to be as little as 2.5 fold lower than clones in the unfiltered library, and also indicated that the P. ginseng genome may contain a large fraction of methylated repetitive DNA elements. A total of 99 and 100 highly stringent STS primer sets were designed from the filtered and unfiltered library, respectively. Amplification products were tested for latent polymorphism across six cultivars of P. ginseng and other 2 Panax species using six endonucleases recognizing four-bases. STS primer sets described here will be useful for marker-assisted selection, genome mapping and line discrimination of P. ginseng or its cultivars from other Panax species.

Heparin/Heparan Sulfate 6-O-Sulfatase from Flavobacterium heparinum

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

Heparin/Heparan Sulfate N-Sulfamidase from Flavobacterium heparinum

Sulfated polysaccharides such as heparin and heparan sulfate glycosaminoglycans (HSGAGs) are chemically and structurally heterogeneous biopolymers that that function as key regulators of numerous biological functions. The elucidation of HSGAG fine structure is fundamental to understanding their functional diversity, and this is facilitated by the use of select degrading enzymes of defined substrate specificity. Our previous studies have reported the cloning, characterization, recombinant expression, and structure-function analysis in Escherichia coli of the Flavobacterium heparinum 2-O-sulfatase and 6-O-sulfatase enzymes that cleave O-sulfate groups from specific locations of the HSGAG polymer. Building on these preceding studies, we report here the molecular cloning and recombinant expression in Escherichia coli of an N-sulfamidase, specific for HSGAGs. In addition, we examine the basic enzymology of this enzyme through molecular modeling studies and structure-function analysis of substrate specificity and basic biochemistry. We use the results from these studies to propose a novel mechanism for nitrogen-sulfur bond cleavage by the N-sulfamidase. Taken together, our structural and biochemical studies indicate that N-sulfamidase is a predominantly exolytic enzyme that specifically acts on N-sulfated and 6-O-desulfated glucosamines present as monosaccharides or at the nonreducing end of odd-numbered oligosaccharide substrates. In conjunction with the previously reported specificities for the F. heparinum 2-O-sulfatase, 6-O-sulfatase, and unsaturated glucuronyl hydrolase, we are able to now reconstruct in vitro the defined exolytic sequence for the heparin and heparan sulfate degradation pathway of F. heparinum and apply these enzymes in tandem toward the exo-sequencing of heparin-derived oligosaccharides.

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE http://www.epibio.com/item.asp?ID=385). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE http://www.epibio.com/item.asp?ID=385). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

A simple, rapid and efficient method for the extraction of genomic DNA from Allium roseum L. (Alliaceae)

The isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including long range PCR, endonuclease restriction digestion, southern blot analysis, and genomic library construction. Many protocols are available for the extraction of DNA from plant material, but obtain it is difficult in many plants because of metabolites that interfere with DNA isolation procedures and subsequent applications. With frame of the present work, we developed the first reliable and efficient method for isolating Allium roseum L. genomic DNA that is free from polysaccharides and polyphenols. This protocol uses 100 mM Tris-HCl (pH 8), 20 mM EDTA, 1.4 M NaCl, 3% PVP (polyvinylpyrrolidone 40.000), 3% mercaptoethanol, and an incubation at 65°C for 1 h. The purity of isolated genomic DNA was confirmed by spectrophotometric analyses (A260/230 ratio of 1.947, A260/280 of 1.804). DNA was obtained in the amount of 189 Allium roseum L. genomic DNA that is free from polysaccharides and polyphenols. This protocol uses 100 mM Tris-HCl (pH 8), 20 mM EDTA, 1.4 M NaCl, 3% PVP (polyvinylpyrrolidone 40.000), 3% mercaptoethanol, and an incubation at 65°C for 1 h. The purity of isolated genomic DNA was confirmed by spectrophotometric analyses (A260/230 ratio of 1.947, A260/280 of 1.804). DNA was obtained in the amount of 189 Allium roseum L. genomic DNA that is free from polysaccharides and polyphenols. This protocol uses 100 mM Tris-HCl (pH 8), 20 mM EDTA, 1.4 M NaCl, 3% PVP (polyvinylpyrrolidone 40.000), 3% mercaptoethanol, and an incubation at 65°C for 1 h. The purity of isolated genomic DNA was confirmed by spectrophotometric analyses (A260/230 ratio of 1.947, A260/280 of 1.804). DNA was obtained in the amount of 189
g per gram of leaf material, and it proved amenable to restriction digestion.

Construction and Application of a Baculovirus Genomic Library

A random genomic library of the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) was constructed and viral factors were identified by screening the regulator(s) for helicase gene expression. DNAs of 238 library plasmids were used to co-transfect with the reporter plasmid, pHp510-luc, in which the luciferase (luc) gene was driven by the baculovirus helicase promoter. Results showed that eight plasmids of the library strengthened the luciferase activity more than 1000-fold. Sequence analyses revealed that all of the eight plasmids contained an intact ie-1 coding region. To confirm the reliability of the screening library, pHp510-luc was co-transfected with the cloned early gene which revealed that the BmNPV IE-1 was the only early factor that could stimulate the helicase promoter. The function analyses suggested that genome-wide screening factors through the library are powerful means to investigate the transcriptional regulation of dsDNA viruses.

Large‐Scale Discovery of Gene‐Enriched SNPs

Whole‐genome association studies of complex traits in higher eukaryotes require a high density of single nucleotide polymorphism (SNP) markers at genome‐wide coverage. To design high‐throughput, multiplexed SNP genotyping assays, researchers must first discover large numbers of SNPs by extensively resequencing multiple individuals or lines. For SNP discovery approaches using short read‐lengths that next‐generation DNA sequencing technologies offer, the highly repetitive and duplicated nature of large plant genomes presents additional challenges. Here, we describe a genomic library construction procedure that facilitates pyrosequencing of genic and low‐copy regions in plant genomes, and a customized computational pipeline to analyze and assemble short reads (100–200 bp), identify allelic reference sequence comparisons, and call SNPs with a high degree of accuracy. With maize (Zea mays L.) as the test organism in a pilot experiment, the implementation of these methods resulted in the identification of 126,683 putative SNPs between two maize inbred lines at an estimated false discovery rate (FDR) of 15.1%. We estimated rates of false SNP discovery using an internal control, and we validated these FDR rates with an external SNP dataset that was generated using locus‐specific PCR amplification and Sanger sequencing. These results show that this approach has wide applicability for efficiently and accurately detecting gene‐enriched SNPs in large, complex plant genomes.

Bead-probe complex capture a couple of SINE and LINE family from genomes of two closely related species of East Asian cyprinid directly using magnetic separation

BackgroundShort and long interspersed elements (SINEs and LINEs, respectively), two types of retroposons, are active in shaping the architecture of genomes and powerful tools for studies of phylogeny and population biology. Here we developed special protocol to apply biotin-streptavidin bead system into isolation of interspersed repeated sequences rapidly and efficiently, in which SINEs and LINEs were captured directly from digested genomic DNA by hybridization to bead-probe complex in solution instead of traditional strategy including genomic library construction and screening.ResultsA new couple of SINEs and LINEs that shared an almost identical 3'tail was isolated and characterized in silver carp and bighead carp of two closely related species. These SINEs (34 members), designated HAmo SINE family, were little divergent in sequence and flanked by obvious TSD indicated that HAmo SINE was very young family. The copy numbers of this family was estimated to 2 × 105 and 1.7 × 105 per haploid genome by Real-Time qPCR, respectively. The LINEs, identified as the homologs of LINE2 in other fishes, had a conserved primary sequence and secondary structures of the 3'tail region that was almost identical to that of HAmo SINE. These evidences suggest that HAmo SINEs are active and amplified recently utilizing the enzymatic machinery for retroposition of HAmoL2 through the recognition of higher-order structures of the conserved 42-tail region. We analyzed the possible structures of HAmo SINE that lead to successful amplification in genome and then deduced that HAmo SINE, SmaI SINE and FokI SINE that were similar in sequence each other, were probably generated independently and created by LINE family within the same lineage of a LINE phylogeny in the genomes of different hosts.ConclusionThe presented results show the advantage of the novel method for retroposons isolation and a pair of young SINE family and its partner LINE family in two carp fishes, which strengthened the hypotheses containing the slippage model for initiation of reverse transcription, retropositional parasitism of SINEs on LINEs, the formation of the stem loop structure in 3'tail region of some SINEs and LINEs and the mechanism of template switching in generating new SINE family.

Construction of a BAC Library for a Defoliating Insect-Resistant Soybean and Identification of Candidate Clones Using a Novel Approach

Positional cloning of an insect-resistance quantitative trait locus (QTL) requires the construction of a large-insert genomic DNA library from insect-resistant genotypes. To facilitate cloning of a major defoliating insect-resistance QTL on linkage group M of the soybean genetic map, a bacterial artificial chromosome (BAC) library for PI 229358 was constructed and characterized. The HindIII BAC library contains 55,296 clones with an average insert size 131 kb. This library represents a 6-fold soybean haploid genome equivalents, allowing a 99.8% probability of recovering any specific sequence of interest in soybean. BAC filters were screened with a genomic DNA probe Sat_258sc2 obtained through genome walking from flanking sequences of a simple sequence repeat (SSR) marker, Sat_258, which links to the insect-resistance QTL. Thirteen BAC clones were identified positive for Sat_258sc2, and two of them were confirmed to carry Sat_258. The results suggest that this library is useful in positional cloning of the major insect-resistance QTL, and the approach presented here can be used to screen a BAC library for a SSR marker without requiring the creation of BAC pools.

Genetic Toggling of Alkaline Phosphatase Folding Reveals Signal Peptides for All Major Modes of Transport across the Inner Membrane of Bacteria

Prediction of export pathway specificity in prokaryotes is a challenging endeavor due to the similar overall architecture of N-terminal signal peptides for the Sec-, SRP- (signal recognition particle), and Tat (twin arginine translocation)-dependent pathways. Thus, we sought to create a facile experimental strategy for unbiased discovery of pathway specificity conferred by N-terminal signals. Using a limited collection of Escherichia coli strains that allow protein oxidation in the cytoplasm or, conversely, disable protein oxidation in the periplasm, we were able to discriminate the specific mode of export for PhoA (alkaline phosphatase) fusions to signal peptides for all of the major modes of transport across the inner membrane (Sec, SRP, or Tat). Based on these findings, we developed a mini-Tn5 phoA approach to isolate pathway-specific export signals from libraries of random fusions between exported proteins and the phoA gene. Interestingly, we observed that reduced PhoA was exported in a Tat-independent manner when targeted for Tat export in the absence of the essential translocon component TatC. This suggests that initial docking to TatC serves as a key specificity determinant for Tat-specific routing of PhoA, and in its absence, substrates can be rerouted to the Sec pathway, provided they remain compatible with the Sec export mechanism. Finally, the utility of our approach was demonstrated by experimental verification that four secreted proteins from Mycobacterium tuberculosis carrying putative Tat signals are bona fide Tat substrates and thus represent potential Tat-dependent virulence factors in this important human pathogen.

New strategy for Pinus pinaster genomic library construction in bacterial artificial chromosomes

To learn about the structure and organization of plant genomes, molecular cloning of large genomic fragments for representative gene libraries construction is required. The genome library construction on bacterial artificial chromosome (BAC) is one of the most frequently used tools for this purpose, and the chosen strategy in this work. But when working with species containing very large genomes, such as pine, construction of this kind of gene libraries is a laborious and expensive task. In this study we have developed a method that starts from P. pinaster cotyledons to construct a pooled BAC library that drastically reduces cost, space and time required to clone a genotype of interesThe current BAC library comprises 83 groups of cells with an average of 4000 clones per group and accounts for a 0.8 X genome content of P. pinaster. It is also shown that a BAC of interest can be quickly identified by PCR (polymerase chain reaction): a pool containing a BAC clone that carries a coding sequence similar to a Class I chitinase of Picea abies has been identified, as well as another BAC clone in a different pool containing a sequence with homology to an unknown P.taeda mRNA. This strategy will allow screening and storing gene libraries from large genome organisms and a quickly location of genes of interest, the method been described in detail.

An Insecticidal GroEL Protein with Chitin Binding Activity from Xenorhabdus nematophila

Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an approximately 58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of approximately 3.6 microg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed alpha-chitin binding activity with Kd approximately 0.64 microm and Bmax approximately 4.68 micromol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an approximately 8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity.

Cloning of glyceraldehyde‐3‐phosphate dehydrogenase from an Antarctic psychrophilic bacterium by inverse and splinkerette PCR

Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterium codenamed pi9 was selected for the cloning of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme in the glycolytic pathway. Here, the cloning of an 1,113 bp fragment of GAPDH gene which covers the 1,002 bp open reading frame by using multiple PCR steps is described. The partial sequence of this gene was PCR amplified by using degenerate primers followed by the cloning of the flanking sequences by inverse and splinkerette PCR techniques. The success in cloning the GAPDH gene by PCR has bypassed the more time consuming genomic library construction and screening method. The full length GAPDH protein was subsequently expressed in E. coli, purified as His-tag protein and confirmed to be catalytically active. This work demonstrated the use of multiple PCR techniques to clone a gene based solely on sequence comparison. It also laid the foundation for further biochemical and structural characterizations of GAPDH from a psychrophilic bacterium by providing a highly purified recombinant protein sample.

Polymorphic nuclear microsatellite loci for studies of brown crab, Cancer pagurus L.

Twelve polymorphic microsatellite DNA loci were isolated from the brown crab, Cancer pagurus L., by construction of microsatellite-enriched genomic libraries. Genotyping of 40 individuals from Norfolk (UK) revealed variable levels of locus polymorphism with an average of 7.75 alleles per locus (range 2-22). The observed and expected heterozygosities per locus ranged from 0.025 to 0.868 and from 0.025 to 0.947, respectively. No evidence of linkage disequilibrium was detected between pairs of loci and genotype proportions at all loci conformed to Hardy-Weinberg equilibrium expectations. The microsatellite loci developed constitute a suite of genetic markers applicable to numerous areas of C. pagurus research.

Development of a Genomic Microsatellite Library in Perennial Ryegrass (Lolium perenne) and its Use in Trait Mapping

Perennial ryegrass (Lolium perenne) is one of the key forage and amenity grasses throughout the world. In the UK it accounts for 70 % of all agricultural land use with an estimated farm gate value of 6 billion pounds per annum. However, in terms of the genetic resources available, L. perenne has lagged behind other major crops in Poaceae. The aim of this project was therefore the construction of a microsatellite-enriched genomic library for L. perenne to increase the number of genetic markers available for both marker-assisted selection in breeding programmes and gene isolation. Primers for 229 non-redundant microsatellite markers were designed and used to screen two L. perenne genotypes, one amenity and one forage. Of the 229 microsatellites, 95 were found to show polymorphism between amenity and forage genotypes. A selection of microsatellite primers was selected from these 95 and used to screen two mapping populations derived from intercrossing and backcrossing the two forage and amenity grass genotypes. The utility of the resulting genetic maps for analysis of the genetic control of target traits was demonstrated by the mapping of genes associated with heading date to linkage groups 4 and 7.

A novel retrieval system for nearly complete microbial genomic fragments from soil samples

The construction of a complex genomic library is one of the comprehensive ways to study a complex bacterial community and to access the variety of metabolic pathways present in the rich soil environment. In this report, we developed a new protocol whereby we are able to retrieve nearly complete microbe genomic fragments from soil samples, which are employed to generate a metagenomic library for visualizing the basic scaffolding of the soil microbial community. The use of direct cell lysis within soil-embedded agarose plugs, along with a double-size selection, enabled us to successfully isolate pure and high-molecular weight DNA (0.1–1 Mb) without the need for any further purification. A metagenomic library containing 1.2 Gbp of DNA in total was constructed. Furthermore, analysis of the microbial community structure using 16S rDNA partial sequences found the dominant phylotypes to consist of α- Proteobacteria and Actinobacteria, which are similar to those seen in forest and agricultural soils, and numerous uncultured microbes from a wide variety of bacterial taxa as well. In conclusion, this study presents a novel protocol for generating a metagenomic library that carries much larger and diverse DNA fragments from soil bacteria that will be applied for the reconstruction of soil microbial genomes and the discovery of novel habitat-specific pathways.

Generation and Screening of a BAC Library from a Diploid Potato Clone to Unravel Durable Late Blight Resistance on Linkage Group IV

We describe the construction and screening of a large insert genomic library from the diploid potato clone HB171(13) that has been shown to express durable quantitative field resistance to Phytophthora infestans, the causal agent of potato late blight disease. Integrated genetic mapping of the field resistance quantitative trait locus with markers developed from populations segregating for Rpi-blb3, Rpi-abpt, R2, and R2-like resistance, all located on linkage group IV, has positioned the field resistance QTL within the proximity of this R gene cluster. The library has been successfully screened with resistance gene analogues (RGA) potentially linked to the R gene cluster. Over 30 positive BAC clones were identified and confirmed by PCR and Southern hybridisations to harbour RGA-like sequences. In addition, BAC end sequencing of positive clones has corroborated two BAC clones with a very high level of nucleotide similarity to the RGA probes utilised.

SAT, a flexible and optimized Web application for SSR marker development

BackgroundSimple Sequence Repeats (SSRs), or microsatellites, are among the most powerful genetic markers known. A common method for the development of SSR markers is the construction of genomic DNA libraries enriched for SSR sequences, followed by DNA sequencing. However, designing optimal SSR markers from bulk sequence data is a laborious and time-consuming process.ResultsSAT (SSR Analysis Tool) is a user-friendly Web application developed to minimize tedious manual operations and reduce errors. This tool facilitates the integration, analysis and display of sequence data from SSR-enriched libraries.SAT is designed to successively perform base calling and quality evaluation of chromatograms, eliminate cloning vector, adaptors and low quality sequences, detect chimera or partially digested sequences, search for SSR motifs, cluster and assemble the redundant sequences, and design SSR primer pairs. An additional virtual PCR step establishes primer specificity. Users may modify the different parameters of each step of the SAT analysis.Although certain steps are compulsory, such as SSR motifs search and sequence assembly, users do not have to run the entire pipeline, and they can choose selectively which steps to perform. A database allows users to store and query results, and to redo individual steps of the workflow.ConclusionThe SAT Web application is available at , and a standalone command-line version is also freely downloadable. Users must send an email to the SAT administrator tropgene@cirad.fr to request a login and password.