Large scale single nucleotide polymorphism discovery in unsequenced genomes using second generation high throughput sequencing technology: applied to turkey

<p>Abstract</p> <p>Background</p> <p>The development of second generation sequencing methods has enabled large scale DNA variation studies at moderate cost. For the high throughput discovery of single nucleotide polymorphisms (SNPs) in species lacking a sequenced reference genome, we set-up an analysis pipeline based on a short read de novo sequence assembler and a program designed to identify variation within short reads. To illustrate the potential of this technique, we present the results obtained with a randomly sheared, enzymatically generated, 2-3 kbp genome fraction of six pooled <it>Meleagris gallopavo </it>(turkey) individuals.</p> <p>Results</p> <p>A total of 100 million 36 bp reads were generated, representing approximately 5-6% (~62 Mbp) of the turkey genome, with an estimated sequence depth of 58. Reads consisting of bases called with less than 1% error probability were selected and assembled into contigs. Subsequently, high throughput discovery of nucleotide variation was performed using sequences with more than 90% reliability by using the assembled contigs that were 50 bp or longer as the reference sequence. We identified more than 7,500 SNPs with a high probability of representing true nucleotide variation in turkeys. Increasing the reference genome by adding publicly available turkey BAC-end sequences increased the number of SNPs to over 11,000. A comparison with the sequenced chicken genome indicated that the assembled turkey contigs were distributed uniformly across the turkey genome. Genotyping of a representative sample of 340 SNPs resulted in a SNP conversion rate of 95%. The correlation of the minor allele count (MAC) and observed minor allele frequency (MAF) for the validated SNPs was 0.69.</p> <p>Conclusion</p> <p>We provide an efficient and cost-effective approach for the identification of thousands of high quality SNPs in species currently lacking a sequenced genome and applied this to turkey. The methodology addresses a random fraction of the genome, resulting in an even distribution of SNPs across the targeted genome.</p

The Complete Chloroplast Genome of 17 Individuals of Pest Species Jacobaea vulgaris: SNPs, Microsatellites and Barcoding Markers for Population and Phylogenetic Studies

Invasive individuals from the pest species Jacobaea vulgaris show different allocation patterns in defence and growth compared with native individuals. To examine if these changes are caused by fast evolution, it is necessary to identify native source populations and compare these with invasive populations. For this purpose, we are in need of intraspecific polymorphic markers. We therefore sequenced the complete chloroplast genomes of 12 native and 5 invasive individuals of J. vulgaris with next generation sequencing and discovered single-nucleotide polymorphisms (SNPs) and microsatellites. This is the first study in which the chloroplast genome of that many individuals within a single species was sequenced. Thirty-two SNPs and 34 microsatellite regions were found. For none of the individuals, differences were found between the inverted repeats. Furthermore, being the first chloroplast genome sequenced in the Senecioneae clade, we compared it with four other members of the Asteraceae family to identify new regions for phylogentic inference within this clade and also within the Asteraceae family. Five markers (ndhC-trnV, ndhC-atpE, rps18-rpl20, clpP and psbM-trnD) contained parsimony-informative characters higher than 2%. Finally, we compared two procedures of preparing chloroplast DNA for next generation sequencing

The Use of Flagella and Motility for Plant Colonization and Fitness by Different Strains of the Foodborne Pathogen Listeria monocytogenes

The role of flagella and motility in the attachment of the foodborne pathogen Listeria monocytogenes to various surfaces is mixed with some systems requiring flagella for an interaction and others needing only motility for cells to get to the surface. In nature this bacterium is a saprophyte and contaminated produce is an avenue for infection. Previous studies have documented the ability of this organism to attach to and colonize plant tissue. Motility mutants were generated in three wild type strains of L. monocytogenes by deleting either flaA, the gene encoding flagellin, or motAB, genes encoding part of the flagellar motor, and tested for both the ability to colonize sprouts and for the fitness of that colonization. The motAB mutants were not affected in the colonization of alfalfa, radish, and broccoli sprouts; however, some of the flaA mutants showed reduced colonization ability. The best colonizing wild type strain was reduced in colonization on all three sprout types as a result of a flaA deletion. A mutant in another background was only affected on alfalfa. The third, a poor alfalfa colonizer was not affected in colonization ability by any of the deletions. Fitness of colonization was measured in experiments of competition between mixtures of mutant and parent strains on sprouts. Here the flaA and motAB mutants of the three strain backgrounds were impaired in fitness of colonization of alfalfa and radish sprouts, and one strain background showed reduced fitness of both mutant types on broccoli sprouts. Together these data indicate a role for flagella for some strains to physically colonize some plants, while the fitness of that colonization is positively affected by motility in almost all cases

Genomic analysis and temperature-dependent transcriptome profiles of the rhizosphere originating strain Pseudomonas aeruginosa M18

<p>Abstract</p> <p>Background</p> <p>Our previously published reports have described an effective biocontrol agent named <it>Pseudomonas </it>sp. M18 as its 16S rDNA sequence and several regulator genes share homologous sequences with those of <it>P. aeruginosa</it>, but there are several unusual phenotypic features. This study aims to explore its strain specific genomic features and gene expression patterns at different temperatures.</p> <p>Results</p> <p>The complete M18 genome is composed of a single chromosome of 6,327,754 base pairs containing 5684 open reading frames. Seven genomic islands, including two novel prophages and five specific non-phage islands were identified besides the conserved <it>P. aeruginosa </it>core genome. Each prophage contains a putative chitinase coding gene, and the prophage II contains a <it>capB </it>gene encoding a putative cold stress protein. The non-phage genomic islands contain genes responsible for pyoluteorin biosynthesis, environmental substance degradation and type I and III restriction-modification systems. Compared with other <it>P. aeruginosa </it>strains, the fewest number (3) of insertion sequences and the most number (3) of clustered regularly interspaced short palindromic repeats in M18 genome may contribute to the relative genome stability. Although the M18 genome is most closely related to that of <it>P. aeruginosa </it>strain LESB58, the strain M18 is more susceptible to several antimicrobial agents and easier to be erased in a mouse acute lung infection model than the strain LESB58. The whole M18 transcriptomic analysis indicated that 10.6% of the expressed genes are temperature-dependent, with 22 genes up-regulated at 28°C in three non-phage genomic islands and one prophage but none at 37°C.</p> <p>Conclusions</p> <p>The <it>P. aeruginosa </it>strain M18 has evolved its specific genomic structures and temperature dependent expression patterns to meet the requirement of its fitness and competitiveness under selective pressures imposed on the strain in rhizosphere niche.</p

Combined genome and transcriptome sequencing to investigate the plant cell wall degrading enzyme system in the thermophilic fungus Malbranchea cinnamomea

Background: Genome and transcriptome sequencing has greatly facilitated the understanding of biomass-degrading mechanisms in a number of fungal species. The information obtained enables the investigation and discovery of genes encoding proteins involved in plant cell wall degradation, which are crucial for saccharification of lignocellulosic biomass in second-generation biorefinery applications. The thermophilic fungus Malbranchea cinnamomea is an efficient producer of many industrially relevant enzymes and a detailed analysis of its genomic content will considerably enhance our understanding of its lignocellulolytic system and promote the discovery of novel proteins. Results: The 25-million-base-pair genome of M. cinnamomea FCH 10.5 was sequenced with 225x coverage. A total of 9437 protein-coding genes were predicted and annotated, among which 301 carbohydrate-active enzyme (CAZyme) domains were found. The putative CAZymes of M. cinnamomea cover cellulases, hemicellulases, chitinases and pectinases, equipping the fungus with the ability to grow on a wide variety of biomass types. Upregulation of 438 and 150 genes during growth on wheat bran and xylan, respectively, in comparison to growth on glucose was revealed. Among the most highly upregulated CAZymes on xylan were glycoside hydrolase family GH10 and GH11 xylanases, as well as a putative glucuronoyl esterase and a putative lytic polysaccharide monooxygenase (LPMO). AA9-domain-containing proteins were also found to be upregulated on wheat bran, as well as a putative cutinase and a protein harbouring a CBM9 domain. Several genes encoding secreted proteins of unknown function were also more abundant on wheat bran and xylan than on glucose. Conclusions: The comprehensive combined genome and transcriptome analysis of M. cinnamomea provides a detailed insight into its response to growth on different types of biomass. In addition, the study facilitates the further exploration and exploitation of the repertoire of industrially relevant lignocellulolytic enzymes of this fungus