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The duplicated and the highly repetitive nature of the maize genome has historically impeded the development of true single nucleotide polymorphism (SNP) markers in this crop. Recent advances in genome complexity reduction methods coupled with sequencing-by-synthesis technologies permit the implementation of efficient genome-wide SNP discovery in maize. In this study, we have applied Complexity Reduction of Polymorphic Sequences technology (Keygene N.V., Wageningen, The Netherlands) for the identification of informative SNPs between two genetically distinct maize inbred lines of North and South American origins. This approach resulted in the discovery of 1,123 putative SNPs representing low and single copy loci. In silico and experimental (Illumina GoldenGate (GG) assay) validation of putative SNPs resulted in mapping of 604 markers, out of which 188 SNPs represented 43 haplotype blocks distributed across all ten chromosomes. We have determined and clearly stated a specific combination of stringent criteria (>0.3 minor allele frequency, >0.8 GenTrainScore and >0.5 Chi_test100 score) necessary for the identification of highly polymorphic and genetically stable SNP markers. Due to these criteria, we identified a subset of 120 high-quality SNP markers to leverage in GG assay-based marker-assisted selection projects. A total of 32 high-quality SNPs represented 21 haplotypes out of 43 identified in this study. The information on the selection criteria of highly polymorphic SNPs in a complex genome such as maize and the public availability of these SNP assays will be of great value for the maize molecular genetics and breeding community.

Summary of the genes found in the genetic recombination regions in clade SC2-WA. Genes present in the regions with recombination events in each clade are summarised. (DOCX 124Â kb)

Distribution of the accessory genes and antibiotic resistance conferring elements in the ST217 isolates. a) Maximum likelihood phylogeny of the isolates rooted using isolates from ST615 as an outgroup (not shown). b) Number of shared accessory open reading frames (ORFs) or genes between pairs of isolates in the phylogeny on the left side and at the top. c) Colour strips showing the continent and country of origin of the isolates and their SCs. d) A heatmap showing the number of shared accessory genes between each pair of isolates in the phylogeny (panel [a] and [e]). (PDF 4076Â kb)

Recombination events identified in the South East African clade (SC3-SEA). (a) Maximum likelihood phylogeny showing genetic relationship of the isolates in the SC. Color strips labeled (b) and (c) shows country and continent of origin of the isolates respectively. (d) Annotations in the reference serotype 1 genome. (e) Horizontal tracks from the phylogenetic tips represent each genome. The coloured blocks shows locations of the recombination events in the chromosome. Red blocks show shared recombination events identified in at least two isolate while the blue blocks show strain specific (unique) recombination events. (PDF 1077Â kb)

Summary of the genes found in the genetic recombination regions in clade SC1-SA. Genes present in the regions with recombination events in each clade are summarised. (DOCX 85Â kb)

Recombination events identified in the West African clade (SC2-WA). (a) Maximum likelihood phylogeny showing genetic relationship of the isolates in the SC. Color strips labeled (b) and (c) shows country and continent of origin of the isolates respectively. (d) Annotations in the reference serotype 1 genome. (e) Horizontal tracks from the phylogenetic tips represent each genome. The coloured blocks shows locations of the recombination events in the chromosome. Red blocks show shared recombination events identified in at least two isolate while the blue blocks show strain specific (unique) recombination events. (PDF 1452Â kb)

Estimated evolutionary parameters for all the clades. Each parameter estimate was calculated using MEGA. (DOC 80Â kb)

Summary of the genes found in the genetic recombination regions in clade SC3-SEA. Genes present in the regions with recombination events in each clade are summarised. (DOCX 133Â kb)

Clade specific phylogenies showing country of origin of the ST217 isolates. Maximum likelihood phylogenetic trees for clade (a) SC1-SA, (b) SC2-WA and (c) SC3-SEA. (PDF 292Â kb)

Recombination events identified in the South African clade (SC1-SA). (a) Maximum likelihood phylogeny showing genetic relationship of the isolates in the SC. Color strips labeled (b) and (c) shows country and continent of origin of the isolates respectively. (d) Annotations in the reference serotype 1 genome. (e) Horizontal tracks from the phylogenetic tips represent each genome. The coloured blocks shows locations of the recombination events in the chromosome. Red blocks show shared recombination events identified in at least two isolate while the blue blocks show strain specific (unique) recombination events. (PDF 641Â kb)

International audience; Serotype 1 Streptococcus pneumoniae is a leading cause of invasive pneumococcal disease (IPD) worldwide, with the highest burden in developing countries. We report the whole-genome sequencing analysis of 448 serotype 1 isolates from 27 countries worldwide (including 11 in Africa). The global serotype 1 population shows a strong phylogeographic structure at the continental level, and within Africa there is further region-specific structure. Our results demonstrate that region-specific diversification within Africa has been driven by limited cross-region transfer events, genetic recombination and antimicrobial selective pressure. Clonal replacement of the dominant serotype 1 clones circulating within regions is uncommon; however, here we report on the accessory gene content that has contributed to a rare clonal replacement event of ST3081 with ST618 as the dominant cause of IPD in the Gambia.

Streptococcus pneumoniae is a nasopharyngeal commensal that occasionally invades normally sterile sites to cause bloodstream infection and meningitis. Although the pneumococcal population structure and evolutionary genetics are well defined, it is not clear whether pneumococci that cause meningitis are genetically distinct from those that do not. Here, we used whole-genome sequencing of 140 isolates of S. pneumoniae recovered from bloodstream infection ( n = 70) and meningitis ( n = 70) to compare their genetic contents. By fitting a double-exponential decaying-function model, we show that these isolates share a core of 1,427 genes (95% confidence interval [CI], 1,425 to 1,435 genes) and that there is no difference in the core genome or accessory gene content from these disease manifestations. Gene presence/absence alone therefore does not explain the virulence behavior of pneumococci that reach the meninges. Our analysis, however, supports the requirement of a range of previously described virulence factors and vaccine candidates for both meningitis- and bacteremia-causing pneumococci. This high-resolution view suggests that, despite considerable competency for genetic exchange, all pneumococci are under considerable pressure to retain key components advantageous for colonization and transmission and that these components are essential for access to and survival in sterile sites.

Serotype 1 Streptococcus pneumoniae is a leading cause of invasive pneumococcal disease (IPD) worldwide, with the highest burden in developing countries. We report the whole-genome sequencing analysis of 448 serotype 1 isolates from 27 countries worldwide (including 11 in Africa). The global serotype 1 population shows a strong phylogeographic structure at the continental level, and within Africa there is further region-specific structure. Our results demonstrate that region-specific diversification within Africa has been driven by limited cross-region transfer events, genetic recombination and antimicrobial selective pressure. Clonal replacement of the dominant serotype 1 clones circulating within regions is uncommon; however, here we report on the accessory gene content that has contributed to a rare clonal replacement event of ST3081 with ST618 as the dominant cause of IPD in the Gambia.

We present whole genome profiling (WGP), a novel next-generation sequencing-based physical mapping technology for construction of bacterial artificial chromosome (BAC) contigs of complex genomes, using Arabidopsis thaliana as an example. WGP leverages short read sequences derived from restriction fragments of two-dimensionally pooled BAC clones to generate sequence tags. These sequence tags are assigned to individual BAC clones, followed by assembly of BAC contigs based on shared regions containing identical sequence tags. Following in silico analysis of WGP sequence tags and simulation of a map of Arabidopsis chromosome 4 and maize, a WGP map of Arabidopsis thaliana ecotype Columbia was constructed de novo using a six-genome equivalent BAC library. Validation of the WGP map using the Columbia reference sequence confirmed that 350 BAC contigs (98%) were assembled correctly, spanning 97% of the 102-Mb calculated genome coverage. We demonstrate that WGP maps can also be generated for more complex plant genomes and will serve as excellent scaffolds to anchor genetic linkage maps and integrate whole genome sequence data.

Application of single nucleotide polymorphisms (SNPs) is revolutionizing human bio-medical research. However, discovery of polymorphisms in low polymorphic species is still a challenging and costly endeavor, despite widespread availability of Sanger sequencing technology. We present CRoPS™ as a novel approach for polymorphism discovery by combining the power of reproducible genome complexity reduction of AFLP® with Genome Sequencer (GS) 20/GS FLX next-generation sequencing technology. With CRoPS, hundreds-of-thousands of sequence reads derived from complexity-reduced genome sequences of two or more samples are processed and mined for SNPs using a fully-automated bioinformatics pipeline. We show that over 75% of putative maize SNPs discovered using CRoPS are successfully converted to SNPWave® assays, confirming them to be true SNPs derived from unique (single-copy) genome sequences. By using CRoPS, polymorphism discovery will become affordable in organisms with high levels of repetitive DNA in the genome and/or low levels of polymorphism in the (breeding) germplasm without the need for prior sequence information.

Volume 83, no. 10, p. [4165–4173][1], 2015. Page 4165, abstract, line 5, “1,427” should read “1,428.” Page 4167, column 1, line 40: “1,427” should read “1,428.” Page 4171, column 2, line 1: “1,427” should read “1,428.” [1]: /lookup/doi/10.1128/IAI.00814-15

Characteristics and assembly statistics for the ST217 study isolates. (DOCX 166Â kb)

Characteristics and assembly statistics for the ST217 study isolates. (DOCX 166Â kb)

Distribution of genes associated with virulence and colonisation in the ST217 in serotype 1 isolates. Presence of the genes was screened in all the isolates using BLAST as described in the methods section. (PDF 1702Â kb)

Deletion of the immunoglobulin A (iga) protease gene in the ST217 isolates. Genomic comparison of one of the serotype 1 isolates (ERS194295) that lacked the iga gene against the TIGR4 reference S. pneumoniae genome with an intact iga gene in its chromosome to determine the location and structure of the genomic deletion in the ST217 isolates. Sequence comparison was performed by BLASTN and visualised with Artemis Comparison Tool (ACT). (PDF 528Â kb)

Intragenic number of observed and expected numbers of SNPs in the ST217 isolates. All the genes in the ST217 isolates, sizes, observed (OSNP) and expected (ESNP) number of SNPs, ratio OSNP to ESNP and products of each gene are summarised. (DOCX 315Â kb)

Deletion of the immunoglobulin A (iga) protease gene in the ST217 isolates. Genomic comparison of one of the serotype 1 isolates (ERS194295) that lacked the iga gene against the TIGR4 reference S. pneumoniae genome with an intact iga gene in its chromosome to determine the location and structure of the genomic deletion in the ST217 isolates. Sequence comparison was performed by BLASTN and visualised with Artemis Comparison Tool (ACT). (PDF 528Â kb)

Intragenic number of observed and expected numbers of SNPs in the ST217 isolates. All the genes in the ST217 isolates, sizes, observed (OSNP) and expected (ESNP) number of SNPs, ratio OSNP to ESNP and products of each gene are summarised. (DOCX 315Â kb)