A hybrid BAC physical map of potato: a framework for sequencing a heterozygous genome

<p>Abstract</p> <p>Background</p> <p>Potato is the world's third most important food crop, yet cultivar improvement and genomic research in general remain difficult because of the heterozygous and tetraploid nature of its genome. The development of physical map resources that can facilitate genomic analyses in potato has so far been very limited. Here we present the methods of construction and the general statistics of the first two genome-wide BAC physical maps of potato, which were made from the heterozygous diploid clone RH89-039-16 (RH).</p> <p>Results</p> <p>First, a gel electrophoresis-based physical map was made by AFLP fingerprinting of 64478 BAC clones, which were aligned into 4150 contigs with an estimated total length of 1361 Mb. Screening of BAC pools, followed by the KeyMaps <it>in silico </it>anchoring procedure, identified 1725 AFLP markers in the physical map, and 1252 BAC contigs were anchored the ultradense potato genetic map. A second, sequence-tag-based physical map was constructed from 65919 whole genome profiling (WGP) BAC fingerprints and these were aligned into 3601 BAC contigs spanning 1396 Mb. The 39733 BAC clones that overlap between both physical maps provided anchors to 1127 contigs in the WGP physical map, and reduced the number of contigs to around 2800 in each map separately. Both physical maps were 1.64 times longer than the 850 Mb potato genome. Genome heterozygosity and incomplete merging of BAC contigs are two factors that can explain this map inflation. The contig information of both physical maps was united in a single table that describes hybrid potato physical map.</p> <p>Conclusions</p> <p>The AFLP physical map has already been used by the Potato Genome Sequencing Consortium for sequencing 10% of the heterozygous genome of clone RH on a BAC-by-BAC basis. By layering a new WGP physical map on top of the AFLP physical map, a genetically anchored genome-wide framework of 322434 sequence tags has been created. This reference framework can be used for anchoring and ordering of genomic sequences of clone RH (and other potato genotypes), and opens the possibility to finish sequencing of the RH genome in a more efficient way via high throughput next generation approaches.</p

Helping hands: A cluster randomised trial to evaluate the effectiveness of two different strategies for promoting hand hygiene in hospital nurses

Background: hand hygiene prescriptions are the most important measure in the prevention of hospital-acquired infections. Yet, compliance rates are generally below 50% of all opportunities for hand hygiene. This study aims at evaluating the short- and long-term effects of two different strategies for promoting hand hygiene in hospital nurses.Methods/design: this study is a cluster randomised controlled trial with inpatient wards as the unit of randomisation. Guidelines for hand hygiene will be implemented in this study. Two strategies will be used to improve the adherence to guidelines for hand hygiene. The state-of-the-art strategy is derived from the literature and includes education, reminders, feedback, and targeting adequate products and facilities. The extended strategy also contains activities aimed at influencing social influence in groups and enhancing leadership. The unique contribution of the extended strategy is built upon relevant behavioural science theories. The extended strategy includes all elements of the state-of-the-art strategy supplemented with gaining active commitment and initiative of ward management, modelling by informal leaders at the ward, and setting norms and targets within the team. Data will be collected at four points in time, with six-month intervals. An average of 3,000 opportunities for hand hygiene in approximately 900 nurses will be observed at each time point.Discussion: performing and evaluating an implementation strategy that also targets the social context of teams may considerably add to the general body of knowledge in this field. Results from our study will allow us to draw conclusions on the effects of different strategies for the implementation of hand hygiene guidelines, and based on these results we will be able to define a preferred implementation strategy for hospital based nursing.Trial registration: the study is registered as a Clinical Trial in ClinicalTrials.gov, dossier number: NCT0054801

A genome-wide genetic map of NB-LRR disease resistance loci in potato

Like all plants, potato has evolved a surveillance system consisting of a large array of genes encoding for immune receptors that confer resistance to pathogens and pests. The majority of these so-called resistance or R proteins belong to the super-family that harbour a nucleotide binding and a leucine-rich-repeat domain (NB-LRR). Here, sequence information of the conserved NB domain was used to investigate the genome-wide genetic distribution of the NB-LRR resistance gene loci in potato. We analysed the sequences of 288 unique BAC clones selected using filter hybridisation screening of a BAC library of the diploid potato clone RH89-039-16 (S. tuberosum ssp. tuberosum) and a physical map of this BAC library. This resulted in the identification of 738 partial and full-length NB-LRR sequences. Based on homology of these sequences with known resistance genes, 280 and 448 sequences were classified as TIR-NB-LRR (TNL) and CC-NB-LRR (CNL) sequences, respectively. Genetic mapping revealed the presence of 15 TNL and 32 CNL loci. Thirty-six are novel, while three TNL loci and eight CNL loci are syntenic with previously identified functional resistance genes. The genetic map was complemented with 68 universal CAPS markers and 82 disease resistance trait loci described in literature, providing an excellent template for genetic studies and applied research in potato

A Next-Generation Sequencing Method for Genotyping-by-Sequencing of Highly Heterozygous Autotetraploid Potato

<div><p>Assessment of genomic DNA sequence variation and genotype calling in autotetraploids implies the ability to distinguish among five possible alternative allele copy number states. This study demonstrates the accuracy of genotyping-by-sequencing (GBS) of a large collection of autotetraploid potato cultivars using next-generation sequencing. It is still costly to reach sufficient read depths on a genome wide scale, across the cultivated gene pool. Therefore, we enriched cultivar-specific DNA sequencing libraries using an in-solution hybridisation method (SureSelect). This complexity reduction allowed to confine our study to 807 target genes distributed across the genomes of 83 tetraploid cultivars and one reference (DM 1–3 511). Indexed sequencing libraries were paired-end sequenced in 7 pools of 12 samples using Illumina HiSeq2000. After filtering and processing the raw sequence data, 12.4 Gigabases of high-quality sequence data was obtained, which mapped to 2.1 Mb of the potato reference genome, with a median average read depth of 63× per cultivar. We detected 129,156 sequence variants and genotyped the allele copy number of each variant for every cultivar. In this cultivar panel a variant density of 1 SNP/24 bp in exons and 1 SNP/15 bp in introns was obtained. The average minor allele frequency (MAF) of a variant was 0.14. Potato germplasm displayed a large number of relatively rare variants and/or haplotypes, with 61% of the variants having a MAF below 0.05. A very high average nucleotide diversity (π = 0.0107) was observed. Nucleotide diversity varied among potato chromosomes. Several genes under selection were identified. Genotyping-by-sequencing results, with allele copy number estimates, were validated with a KASP genotyping assay. This validation showed that read depths of ∼60–80× can be used as a lower boundary for reliable assessment of allele copy number of sequence variants in autotetraploids. Genotypic data were associated with traits, and alleles strongly influencing maturity and flesh colour were identified.</p></div

Mapping of resequencing targets onto the potato genome.

<p>Physical position of the approximately 800 target genes scattered across the 12 potato chromosomes. Physical positions are ordered according to the latest pseudomolecule order (v.3 beta). The distribution of the ∼800 genes reflect the physical proportions of gene rich chromosome arms and gene poor heterochromatic regions.</p

Summary of target enrichment sequence coverage for 84 potato cultivars.

<p>Summary of target enrichment sequence coverage for 84 potato cultivars.</p

Manhattan plots of <i>p</i> values for associations between DNA sequence variants and two phenotypic traits in potato: (A) plant maturity and (B) tuber flesh colour.

<p>The FDR corrected −log₁₀(<i>p</i>) values from GWAS analysis are plotted relative to the physical position on each of 12 potato chromosomes. The horizontal dashed line is plotted at the FDR-corrected significance threshold of α = 0.001 (−log₁₀(<i>p</i>) = 3).</p