Publications by authors named "Hans de Jong"

65 Publications

Commercialized Blood-, Urinary- and Tissue-Based Biomarker Tests for Prostate Cancer Diagnosis and Prognosis.

Cancers (Basel) 2020 Dec 16;12(12). Epub 2020 Dec 16.

Department of Urology, Radboud University Medical Centre, 6525 GA Nijmegen, The Netherlands.

In the diagnosis and prognosis of prostate cancer (PCa), the serum prostate-specific antigen test is widely used but is associated with low specificity. Therefore, blood-, urinary- and tissue-based biomarker tests have been developed, intended to be used in the diagnostic and prognostic setting of PCa. This review provides an overview of commercially available biomarker tests developed to be used in several clinical stages of PCa management. In the diagnostic setting, the following tests can help selecting the right patients for initial and/or repeat biopsy: PHI, 4K, MiPS, SelectMDx, ExoDx, Proclarix, ConfirmMDx, PCA3 and PCMT. In the prognostic setting, the Prolaris, OncotypeDx and Decipher test can help in risk-stratification of patients regarding treatment decisions. Following, an overview is provided of the studies available comparing the performance of biomarker tests. However, only a small number of recently published head-to-head comparison studies are available. In contrast, recent research has focused on the use of biomarker tests in relation to the (complementary) use of multiparametric magnetic resonance imaging in PCa diagnosis.
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http://dx.doi.org/10.3390/cancers12123790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7765473PMC
December 2020

Meiotic crossover reduction by virus-induced gene silencing enables the efficient generation of chromosome substitution lines and reverse breeding in Arabidopsis thaliana.

Plant J 2020 12 20;104(5):1437-1452. Epub 2020 Oct 20.

Laboratory of Genetics, Wageningen University & Research, Droevendaalsesteeg 1, Wageningen, 6708 PB, the Netherlands.

Plant breeding applications exploiting meiotic mutant phenotypes (like the increase or decrease of crossover (CO) recombination) have been proposed over the last years. As recessive meiotic mutations in breeding lines may affect fertility or have other pleiotropic effects, transient silencing techniques may be preferred. Reverse breeding is a breeding technique that would benefit from the transient downregulation of CO formation. The technique is essentially the opposite of plant hybridization: a method to extract parental lines from a hybrid. The method can also be used to efficiently generate chromosome substitution lines (CSLs). For successful reverse breeding, the two homologous chromosome sets of a heterozygous plant must be divided over two haploid complements, which can be achieved by the suppression of meiotic CO recombination and the subsequent production of doubled haploid plants. Here we show the feasibility of transiently reducing CO formation using virus-induced gene silencing (VIGS) by targeting the meiotic gene MSH5 in a wild-type heterozygote of Arabidopsis thaliana. The application of VIGS (rather than using lengthy stable transformation) generates transgene-free offspring with the desired genetic composition: we obtained parental lines from a wild-type heterozygous F in two generations. In addition, we obtained 20 (of the 32 possible) CSLs in one experiment. Our results demonstrate that meiosis can be modulated at will in A. thaliana to generate CSLs and parental lines rapidly for hybrid breeding. Furthermore, we illustrate how the modification of meiosis using VIGS can open routes to develop efficient plant breeding strategies.
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http://dx.doi.org/10.1111/tpj.14990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756339PMC
December 2020

Genetic mapping of Fusarium wilt resistance in a wild banana Musa acuminata ssp. malaccensis accession.

Theor Appl Genet 2020 Dec 12;133(12):3409-3418. Epub 2020 Sep 12.

Laboratory of Phytopathology, Wageningen University and Research, P.O. Box 16, 6700 AA, Wageningen, The Netherlands.

Banana is an important fruit and food crop, but is threatened by Fusarium wilt, one of the most devastating soil-borne fungal diseases. Only host resistance facilitates banana cultivation in infested soils around the world, but the genetic basis of Fusarium wilt of banana (FWB) is unknown. We selfed a heterozygous wild banana accession Musa acuminata ssp. malaccensis (Mam, AA, 2n = 22) to generate a mapping population and to investigate the inheritance of resistance to Race 1 and tropical race 4 (TR4) that cause FWB. Phenotyping (N = 217) revealed segregation for resistance, and genotyping by sequencing resulted in 2802 high-quality single-nucleotide polymorphic markers (SNPs) that were used for genetic mapping. Combined analyses of these data showed that a single dominant resistance locus controls resistance to Race 1 and maps near the distal part of chromosome 10. Recombinants, together with the position of the putative resistance gene, were further analysed using graphical genotyping, which retrieved markers flanking a 360 kb genetic region that associates with Race 1 resistance. The region contains 165 putative genes on the reference genome, including 19 leucine-rich repeat receptor-like kinase-like genes. At the same position and phase, we also identified a QTL for TR4 resistance, showing that the locus for resistance against Race 1 provided partial resistance to TR4. However, this effect was far less significant and hence not included in the mapping. These data support the breeding of new banana varieties with resistance to Fusarium wilt.
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http://dx.doi.org/10.1007/s00122-020-03677-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567712PMC
December 2020

Meiotic recombination profiling of interspecific hybrid F1 tomato pollen by linked read sequencing.

Plant J 2020 05 22;102(3):480-492. Epub 2020 Jan 22.

Business Unit of Bioscience, Cluster Applied Bioinformatics, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.

Genome wide screening of pooled pollen samples from a single interspecific F1 hybrid obtained from a cross between tomato, Solanum lycopersicum and its wild relative, Solanum pimpinellifolium using linked read sequencing of the haploid nuclei, allowed profiling of the crossover (CO) and gene conversion (GC) landscape. We observed a striking overlap between cold regions of CO in the male gametes and our previously established F6 recombinant inbred lines (RILs) population. COs were overrepresented in non-coding regions in the gene promoter and 5'UTR regions of genes. Poly-A/T and AT rich motifs were found enriched in 1 kb promoter regions flanking the CO sites. Non-crossover associated allelic and ectopic GCs were detected in most chromosomes, confirming that besides CO, GC represents also a source for genetic diversity and genome plasticity in tomato. Furthermore, we identified processed break junctions pointing at the involvement of both homology directed and non-homology directed repair pathways, suggesting a recombination machinery in tomato that is more complex than currently anticipated.
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http://dx.doi.org/10.1111/tpj.14640DOI Listing
May 2020

Comparative analysis of repetitive sequences among species from the potato and the tomato clades.

Ann Bot 2019 02;123(3):521-532

Laboratorio de Evolución y Domesticación de las Plantas, Facultad de Agronomía, Universidad de la República, Garzón, Montevideo, Uruguay.

Background And Aims: The genus Solanum includes important vegetable crops and their wild relatives. Introgression of their useful traits into elite cultivars requires effective recombination between hom(e)ologues, which is partially determined by genome sequence differentiation. In this study we compared the repetitive genome fractions of wild and cultivated species of the potato and tomato clades in a phylogenetic context.

Methods: Genome skimming followed by a clustering approach was used as implemented in the RepeatExplorer pipeline. Repeat classes were annotated and the sequences of their main domains were compared.

Key Results: Repeat abundance and genome size were correlated and the larger genomes of species in the tomato clade were found to contain a higher proportion of unclassified elements. Families and lineages of repetitive elements were largely conserved between the clades, but their relative proportions differed. The most abundant repeats were Ty3/Gypsy elements. Striking differences in abundance were found in the highly dynamic Ty3/Gypsy Chromoviruses and Ty1/Copia Tork elements. Within the potato clade, early branching Solanum cardiophyllum showed a divergent repeat profile. There were also contrasts between cultivated and wild potatoes, mostly due to satellite amplification in the cultivated species. Interspersed repeat profiles were very similar among potatoes. The repeat profile of Solanum etuberosum was more similar to that of the potato clade.

Conclusions: The repeat profiles in Solanum seem to be very similar despite genome differentiation at the level of collinearity. Removal of transposable elements by unequal recombination may have been responsible for structural rearrangements across the tomato clade. Sequence variability in the tomato clade is congruent with clade-specific amplification of repeats after its divergence from S. etuberosum and potatoes. The low differentiation among potato and its wild relatives at the level of interspersed repeats may explain the difficulty in discriminating their genomes by genomic in situ hybridization techniques.
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http://dx.doi.org/10.1093/aob/mcy186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377101PMC
February 2019

Intact DNA purified from flow-sorted nuclei unlocks the potential of next-generation genome mapping and assembly in species.

MethodsX 2018 10;5:328-336. Epub 2018 Apr 10.

Laboratory of Genetics, Wageningen University & Research, Wageningen, The Netherlands.

Next-generation genome mapping through nanochannels (Bionano optical mapping) of plant genomes brings genome assemblies to the 'nearly-finished' level for reliable and detailed gene annotations and assessment of structural variations. Despite the recent progress in its development, researchers face the technical challenges of obtaining sufficient high molecular weight (HMW) nuclear DNA due to cell walls which are difficult to disrupt and to the presence of cytoplasmic polyphenols and polysaccharides that co-precipitate or are covalently bound to DNA and might cause oxidation and/or affect the access of nicking enzymes to DNA, preventing downstream applications. Here we describe important improvements for obtaining HMW DNA that we tested on crops and wild relatives. The methods that we further elaborated and refined focus on •Improving flexibility of using different tissues as source materials, like fast-growing root tips and young leaves from seedlings or plantlets.•Obtaining nuclei suspensions through either lab homogenizers or by chopping.•Increasing flow sorting efficiency using DAPI (4',6-diamidino-2-phenylindole) and PI (propidium iodide) DNA stains, with different lasers (UV or 488 nm) and sorting platforms such as the FACSAria and FACSVantage flow sorters, thus making it appropriate for more laboratories working on plant genomics. The obtained nuclei are embedded into agarose plugs for processing and isolating uncontaminated HMW DNA, which is a prerequisite for nanochannel-based next-generation optical mapping strategies.
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http://dx.doi.org/10.1016/j.mex.2018.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058011PMC
April 2018

Optimization of Cell Spreading and Image Quality for the Study of Chromosomes in Plant Tissues.

Methods Mol Biol 2017 ;1669:141-158

Laboratory of Genetics, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.

High-quality chromosome images of mitotic and meiotic cell divisions in plant tissues are inextricably connected with the technical control of cell spread preparations. Superb chromosome slides are the best for studying chromosome morphology and making karyotypes; they also are the best start for a successful fluorescent in situ hybridization experiment. In this study, we describe the essentials for fixation, enzymatic digestion, squash, spread, and dropping protocols for clean and well-differentiated nuclei and chromosome complements. In addition, we focus on the use of standard whole image processing for best sharpness, brightness and contrast adjustments, differentiation of heterochromatin/euchromatin, and high dynamic range imaging of big chromosomes. We also explain how to combine transparent layers or spot channels of different fluorescent images for making publication quality, full color photos.
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http://dx.doi.org/10.1007/978-1-4939-7286-9_12DOI Listing
May 2018

Genetic Dissection of Morphometric Traits Reveals That Phytochrome B Affects Nucleus Size and Heterochromatin Organization in .

G3 (Bethesda) 2017 08 7;7(8):2519-2531. Epub 2017 Aug 7.

Molecular Plant Physiology, Institute of Environmental Biology, Utrecht University, 3584 CH, The Netherlands

Microscopically visible chromatin is partitioned into two major components in nuclei. On one hand, chromocenters are conspicuous foci of highly condensed "heterochromatic" domains that contain mostly repeated sequences. On the other hand, less condensed and gene-rich "euchromatin" emanates from these chromocenters. This differentiation, together with the dynamic nature of chromatin compaction in response to developmental and environmental stimuli, makes a powerful system for studying chromatin organization and dynamics. Heterochromatin dynamics can be monitored by measuring the Heterochromatin Index, , the proportion of nuclei displaying well-defined chromocenters, or the DNA fraction of chromocenters (relative heterochromatin fraction). Both measures are composite traits, thus their values represent the sum of effects of various underlying morphometric properties. We exploited genetic variation between natural occurring accessions to determine the genetic basis of individual nucleus and chromocenter morphometric parameters (area, perimeter, density, roundness, and heterogeneity) that together determine chromatin compaction. Our novel reductionist genetic approach revealed quantitative trait loci (QTL) for all measured traits. Genomic colocalization among QTL was limited, which suggests a complex genetic regulation of chromatin compaction. Yet genomic intervals of QTL for nucleus size (area and perimeter) both overlap with a known QTL for heterochromatin compaction that is explained by natural polymorphism in the red/far-red light and temperature receptor Phytochrome B. Mutant analyses and genetic complementation assays show that Phytochrome B is a negative regulator of nucleus size, revealing that perception of climatic conditions by a Phytochrome-mediated hub is a major determinant for coordinating nucleus size and heterochromatin compaction.
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http://dx.doi.org/10.1534/g3.117.043539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555459PMC
August 2017

Collinearity between potato (Solanum tuberosum L.) and wild relatives assessed by comparative cytogenetic mapping.

Genome 2017 Mar 14;60(3):228-240. Epub 2016 Nov 14.

b Laboratory of Genetics, Wageningen University, Droevendaalsesteeg 1, P.O. Box 16, 6708 PB, Wageningen, the Netherlands.

A major bottleneck to introgressive hybridization is the lack of genome collinearity between the donor (alien) genome and the recipient crop genome. Structural differences between the homeologs may create unbalanced segregation of chromosomes or cause linkage drag. To assess large-scale collinearity between potato and two of its wild relatives (Solanum commersonii and Solanum chacoense), we used BAC-FISH mapping of sequences with known positions on the RH potato map. BAC probes could successfully be hybridized to the S. commersonii and S. chachoense pachytene chromosomes, confirming their correspondence with linkage groups in RH potato. Our study shows that the order of BAC signals is conserved. Distances between BAC signals were quantified and compared; some differences found suggest either small-scale rearrangements or reduction/amplification of repeats. We conclude that S. commersonii and S. chacoense are collinear with cultivated Solanum tuberosum on the whole chromosome scale, making these amenable species for efficient introgressive hybridization breeding.
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http://dx.doi.org/10.1139/gen-2016-0150DOI Listing
March 2017

Molecular, genetic and evolutionary analysis of a paracentric inversion in Arabidopsis thaliana.

Plant J 2016 10 13;88(2):159-178. Epub 2016 Sep 13.

Biosystematics Group, Wageningen University, Wageningen, the Netherlands.

Chromosomal inversions can provide windows onto the cytogenetic, molecular, evolutionary and demographic histories of a species. Here we investigate a paracentric 1.17-Mb inversion on chromosome 4 of Arabidopsis thaliana with nucleotide precision of its borders. The inversion is created by Vandal transposon activity, splitting an F-box and relocating a pericentric heterochromatin segment in juxtaposition with euchromatin without affecting the epigenetic landscape. Examination of the RegMap panel and the 1001 Arabidopsis genomes revealed more than 170 inversion accessions in Europe and North America. The SNP patterns revealed historical recombinations from which we infer diverse haplotype patterns, ancient introgression events and phylogenetic relationships. We find a robust association between the inversion and fecundity under drought. We also find linkage disequilibrium between the inverted region and the early flowering Col-FRIGIDA allele. Finally, SNP analysis elucidates the origin of the inversion to South-Eastern Europe approximately 5000 years ago and the FRI-Col allele to North-West Europe, and reveals the spreading of a single haplotype to North America during the 17th to 19th century. The 'American haplotype' was identified from several European localities, potentially due to return migration.
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http://dx.doi.org/10.1111/tpj.13262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113708PMC
October 2016

Detection of High-grade Prostate Cancer Using a Urinary Molecular Biomarker-Based Risk Score.

Eur Urol 2016 11 20;70(5):740-748. Epub 2016 Apr 20.

Department of Urology, Radboud University Medical Center, Nijmegen, The Netherlands. Electronic address:

Background: To reduce overdiagnosis and overtreatment, a test is urgently needed to detect clinically significant prostate cancer (PCa).

Objective: To develop a multimodal model, incorporating previously identified messenger RNA (mRNA) biomarkers and traditional risk factors that could be used to identify patients with high-grade PCa (Gleason score ≥7) on prostate biopsy.

Design, Setting, And Participants: In two prospective multicenter studies, urine was collected for mRNA profiling after digital rectal examination (DRE) and prior to prostate biopsy. The multimodal risk score was developed on a first cohort (n=519) and subsequently validated clinically in an independent cohort (n=386).

Outcome Measurements And Statistical Analysis: The mRNA levels were measured using reverse transcription quantitative polymerase chain reaction. Logistic regression was used to model patient risk and combine risk factors. Models were compared using the area under the curve (AUC) of the receiver operating characteristic, and clinical utility was evaluated with a decision curve analysis (DCA).

Results And Limitations: HOXC6 and DLX1 mRNA levels were shown to be good predictors for the detection of high-grade PCa. The multimodal approach reached an overall AUC of 0.90 (95% confidence interval [CI], 0.85-0.95) in the validation cohort (AUC 0.86 in the training cohort), with the mRNA signature, prostate-specific antigen (PSA) density, and previous cancer-negative prostate biopsies as the strongest, most significant components, in addition to nonsignificant model contributions of PSA, age, and family history. For another model, which included DRE as an additional risk factor, an AUC of 0.86 (95% CI, 0.80-0.92) was obtained (AUC 0.90 in the training cohort). Both models were successfully validated, with no significant change in AUC in the validation cohort, and DCA indicated a strong net benefit and the best reduction in unnecessary biopsies compared with other clinical decision-making tools, such as the Prostate Cancer Prevention Trial risk calculator and the PCA3 assay.

Conclusions: The risk score based on the mRNA liquid biopsy assay combined with traditional clinical risk factors identified men at risk of harboring high-grade PCa and resulted in a better patient risk stratification compared with current methods in clinical practice. Therefore, the risk score could reduce the number of unnecessary prostate biopsies.

Patient Summary: This study evaluated a novel urine-based assay that could be used as a noninvasive diagnostic aid for high-grade prostate cancer (PCa). When results of this assay are combined with traditional clinical risk factors, risk stratification for high-grade PCa and biopsy decision making are improved.
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http://dx.doi.org/10.1016/j.eururo.2016.04.012DOI Listing
November 2016

Cnidaria: fast, reference-free clustering of raw and assembled genome and transcriptome NGS data.

BMC Bioinformatics 2015 Nov 2;16:352. Epub 2015 Nov 2.

Bioinformatics Group, Department of Plant Sciences, Wageningen University, Wageningen, The Netherlands.

Background: Identification of biological specimens is a requirement for a range of applications. Reference-free methods analyse unprocessed sequencing data without relying on prior knowledge, but generally do not scale to arbitrarily large genomes and arbitrarily large phylogenetic distances.

Results: We present Cnidaria, a practical tool for clustering genomic and transcriptomic data with no limitation on genome size or phylogenetic distances. We successfully simultaneously clustered 169 genomic and transcriptomic datasets from 4 kingdoms, achieving 100% identification accuracy at supra-species level and 78% accuracy at the species level.

Conclusion: CNIDARIA allows for fast, resource-efficient comparison and identification of both raw and assembled genome and transcriptome data. This can help answer both fundamental (e.g. in phylogeny, ecological diversity analysis) and practical questions (e.g. sequencing quality control, primer design).
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http://dx.doi.org/10.1186/s12859-015-0806-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4630969PMC
November 2015

Homologues of potato chromosome 5 show variable collinearity in the euchromatin, but dramatic absence of sequence similarity in the pericentromeric heterochromatin.

BMC Genomics 2015 May 10;16:374. Epub 2015 May 10.

Wageningen UR Plant Breeding, Wageningen University and Research Centre, Droevendaalsesteeg 1, 6708PB, Wageningen, The Netherlands.

Background: In flowering plants it has been shown that de novo genome assemblies of different species and genera show a significant drop in the proportion of alignable sequence. Within a plant species, however, it is assumed that different haplotypes of the same chromosome align well. In this paper we have compared three de novo assemblies of potato chromosome 5 and report on the sequence variation and the proportion of sequence that can be aligned.

Results: For the diploid potato clone RH89-039-16 (RH) we produced two linkage phase controlled and haplotype-specific assemblies of chromosome 5 based on BAC-by-BAC sequencing, which were aligned to each other and compared to the 52 Mb chromosome 5 reference sequence of the doubled monoploid clone DM 1-3 516 R44 (DM). We identified 17.0 Mb of non-redundant sequence scaffolds derived from euchromatic regions of RH and 38.4 Mb from the pericentromeric heterochromatin. For 32.7 Mb of the RH sequences the correct position and order on chromosome 5 was determined, using genetic markers, fluorescence in situ hybridisation and alignment to the DM reference genome. This ordered fraction of the RH sequences is situated in the euchromatic arms and in the heterochromatin borders. In the euchromatic regions, the sequence collinearity between the three chromosomal homologs is good, but interruption of collinearity occurs at nine gene clusters. Towards and into the heterochromatin borders, absence of collinearity due to structural variation was more extensive and was caused by hemizygous and poorly aligning regions of up to 450 kb in length. In the most central heterochromatin, a total of 22.7 Mb sequence from both RH haplotypes remained unordered. These RH sequences have very few syntenic regions and represent a non-alignable region between the RH and DM heterochromatin haplotypes of chromosome 5.

Conclusions: Our results show that among homologous potato chromosomes large regions are present with dramatic loss of sequence collinearity. This stresses the need for more de novo reference assemblies in order to capture genome diversity in this crop. The discovery of three highly diverged pericentric heterochromatin haplotypes within one species is a novelty in plant genome analysis. The possible origin and cytogenetic implication of this heterochromatin haplotype diversity are discussed.
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http://dx.doi.org/10.1186/s12864-015-1578-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4470070PMC
May 2015

Karyotype evolution in apomictic Boechera and the origin of the aberrant chromosomes.

Plant J 2015 Jun;82(5):785-93

CEITEC - Central European Institute of Technology, Masaryk University, Brno, CZ-62500, Czech Republic.

Chromosome rearrangements may result in both decrease and increase of chromosome numbers. Here we have used comparative chromosome painting (CCP) to reconstruct the pathways of descending and ascending dysploidy in the genus Boechera (tribe Boechereae, Brassicaceae). We describe the origin and structure of three Boechera genomes and establish the origin of the previously described aberrant Het and Del chromosomes found in Boechera apomicts with euploid (2n = 14) and aneuploid (2n = 15) chromosome number. CCP analysis allowed us to reconstruct the origin of seven chromosomes in sexual B. stricta and apomictic B. divaricarpa from the ancestral karyotype (n = 8) of Brassicaceae lineage I. Whereas three chromosomes (BS4, BS6, and BS7) retained their ancestral structure, five chromosomes were reshuffled by reciprocal translocations to form chromosomes BS1-BS3 and BS5. The reduction of the chromosome number (from x = 8 to x = 7) was accomplished through the inactivation of a paleocentromere on chromosome BS5. In apomictic 2n = 14 plants, CCP identifies the largely heterochromatic chromosome (Het) being one of the BS1 homologues with the expansion of pericentromeric heterochromatin. In apomictic B. polyantha (2n = 15), the Het has undergone a centric fission resulting in two smaller chromosomes - the submetacentric Het' and telocentric Del. Here we show that new chromosomes can be formed by a centric fission and can be fixed in populations due to the apomictic mode of reproduction.
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http://dx.doi.org/10.1111/tpj.12849DOI Listing
June 2015

Identification of a Candidate Gene Panel for the Early Diagnosis of Prostate Cancer.

Clin Cancer Res 2015 Jul 18;21(13):3061-70. Epub 2015 Mar 18.

Radboud University Nijmegen Medical Centre, Department of Urology, Nijmegen, the Netherlands. Noviogendix, Department of Research and Development, Nijmegen, the Netherlands.

Purpose: Serum PSA (sPSA) testing has led to the identification of patients with indolent prostate cancer, and inevitably overtreatment has become a concern. Progensa PCA3 urine testing was shown to improve the diagnosis of prostate cancer, but its diagnostic value for aggressive prostate cancer is limited. Therefore, urinary biomarkers that can be used for prediction of Gleason score ≥7 prostate cancer in biopsies are urgently needed.

Experimental Design: Using gene expression profiling data, 39 prostate cancer biomarkers were identified. After quantitative PCR analysis on tissue specimens and urinary sediments, eight promising biomarkers for the urinary detection of prostate cancer were selected (ONECUT2, HOXC4, HOXC6, DLX1, TDRD1, NKAIN1, MS4A8B, PPFIA2). The hypothesis that biomarker combinations improve the diagnostic value for aggressive prostate cancer was tested on 358 urinary sediments of an intention-to-treat cohort.

Results: A urinary three-gene panel (HOXC6, TDRD1, and DLX1) had higher accuracy [area under the curve (AUC), 0.77; 95% confidence interval (CI), 0.71-0.83] to predict Gleason score ≥7 prostate cancer in biopsies compared with Progensa PCA3 (AUC, 0.68; 95% CI, 0.62-0.75) or sPSA (AUC, 0.72; 95% CI, 0.65-0.78). Combining the three-gene panel with sPSA further improved the predictive accuracy (AUC, 0.81; 95% CI, 0.75-0.86). The accuracy of the three-gene predictive model was maintained in subgroups with low sPSA concentrations.

Conclusions: The urinary three-gene panel (HOXC6, TDRD1, and DLX1) represents a promising tool to identify patients with aggressive prostate cancer, also in those with low sPSA values. The combination of the urinary three-gene panel with sPSA bears great potential for the early diagnosis of patients with clinically significant prostate cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-14-3334DOI Listing
July 2015

Fluorescent in situ hybridization shows DIPLOSPOROUS located on one of the NOR chromosomes in apomictic dandelions (Taraxacum) in the absence of a large hemizygous chromosomal region.

Genome 2014 Nov 9;57(11-12):609-20. Epub 2015 Feb 9.

Laboratory of Genetics, Wageningen University and Research Centre, P.O. Box 309, NL-6700 AH Wageningen, the Netherlands.

Apomixis in dandelions (Taraxacum: Asteraceae) is encoded by two unlinked dominant loci and a third yet undefined genetic factor: diplosporous omission of meiosis (DIPLOSPOROUS, DIP), parthenogenetic embryo development (PARTHENOGENESIS, PAR), and autonomous endosperm formation, respectively. In this study, we determined the chromosomal position of the DIP locus in Taraxacum by using fluorescent in situ hybridization (FISH) with bacterial artificial chromosomes (BACs) that genetically map within 1.2-0.2 cM of DIP. The BACs showed dispersed fluorescent signals, except for S4-BAC 83 that displayed strong unique signals as well. Under stringent blocking of repeats by C0t-DNA fragments, only a few fluorescent foci restricted to defined chromosome regions remained, including one on the nucleolus organizer region (NOR) chromosomes that contains the 45S rDNAs. FISH with S4-BAC 83 alone and optimal blocking showed discrete foci in the middle of the long arm of one of the NOR chromosomes only in triploid and tetraploid diplosporous dandelions, while signals in sexual diploids were lacking. This agrees with the genetic model of a single dose, dominant DIP allele, absent in sexuals. The length of the DIP region is estimated to cover a region of 1-10 Mb. FISH in various accessions of Taraxacum and the apomictic sister species Chondrilla juncea, confirmed the chromosomal position of DIP within Taraxacum but not outside the genus. Our results endorse that, compared to other model apomictic species, expressing either diplospory or apospory, the genome of Taraxacum shows a more similar and less diverged chromosome structure at the DIP locus. The different levels of allele sequence divergence at apomeiosis loci may reflect different terms of asexual reproduction. The association of apomeiosis loci with repetitiveness, dispersed repeats, and retrotransposons commonly observed in apomictic species may imply a functional role of these shared features in apomictic reproduction, as is discussed.
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http://dx.doi.org/10.1139/gen-2014-0143DOI Listing
November 2014

Introgression browser: high-throughput whole-genome SNP visualization.

Plant J 2015 Apr;82(1):174-82

Applied Bioinformatics, Wageningen University and Research Centre (WUR), Wageningen, The Netherlands.

Breeding by introgressive hybridization is a pivotal strategy to broaden the genetic basis of crops. Usually, the desired traits are monitored in consecutive crossing generations by marker-assisted selection, but their analyses fail in chromosome regions where crossover recombinants are rare or not viable. Here, we present the Introgression Browser (iBrowser), a bioinformatics tool aimed at visualizing introgressions at nucleotide or SNP (Single Nucleotide Polymorphisms) accuracy. The software selects homozygous SNPs from Variant Call Format (VCF) information and filters out heterozygous SNPs, multi-nucleotide polymorphisms (MNPs) and insertion-deletions (InDels). For data analysis iBrowser makes use of sliding windows, but if needed it can generate any desired fragmentation pattern through General Feature Format (GFF) information. In an example of tomato (Solanum lycopersicum) accessions we visualize SNP patterns and elucidate both position and boundaries of the introgressions. We also show that our tool is capable of identifying alien DNA in a panel of the closely related S. pimpinellifolium by examining phylogenetic relationships of the introgressed segments in tomato. In a third example, we demonstrate the power of the iBrowser in a panel of 597 Arabidopsis accessions, detecting the boundaries of a SNP-free region around a polymorphic 1.17 Mbp inverted segment on the short arm of chromosome 4. The architecture and functionality of iBrowser makes the software appropriate for a broad set of analyses including SNP mining, genome structure analysis, and pedigree analysis. Its functionality, together with the capability to process large data sets and efficient visualization of sequence variation, makes iBrowser a valuable breeding tool.
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http://dx.doi.org/10.1111/tpj.12800DOI Listing
April 2015

DELAY OF GERMINATION 1 mediates a conserved coat-dormancy mechanism for the temperature- and gibberellin-dependent control of seed germination.

Proc Natl Acad Sci U S A 2014 Aug 11;111(34):E3571-80. Epub 2014 Aug 11.

School of Biological Sciences, Plant Molecular Science and Centre for Systems and Synthetic Biology, Royal Holloway, University of London, Egham, Surrey TW20 0EX, United Kingdom; Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany ASCR & Palacký University, CZ-78371 Olomouc, Czech Republic; and

Seed germination is an important life-cycle transition because it determines subsequent plant survival and reproductive success. To detect optimal spatiotemporal conditions for germination, seeds act as sophisticated environmental sensors integrating information such as ambient temperature. Here we show that the delay of germination 1 (DOG1) gene, known for providing dormancy adaptation to distinct environments, determines the optimal temperature for seed germination. By reciprocal gene-swapping experiments between Brassicaceae species we show that the DOG1-mediated dormancy mechanism is conserved. Biomechanical analyses show that this mechanism regulates the material properties of the endosperm, a seed tissue layer acting as germination barrier to control coat dormancy. We found that DOG1 inhibits the expression of gibberellin (GA)-regulated genes encoding cell-wall remodeling proteins in a temperature-dependent manner. Furthermore we demonstrate that DOG1 causes temperature-dependent alterations in the seed GA metabolism. These alterations in hormone metabolism are brought about by the temperature-dependent differential expression of genes encoding key enzymes of the GA biosynthetic pathway. These effects of DOG1 lead to a temperature-dependent control of endosperm weakening and determine the optimal temperature for germination. The conserved DOG1-mediated coat-dormancy mechanism provides a highly adaptable temperature-sensing mechanism to control the timing of germination.
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http://dx.doi.org/10.1073/pnas.1403851111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151772PMC
August 2014

Chromosomal organizations of major repeat families on potato (Solanum tuberosum) and further exploring in its sequenced genome.

Mol Genet Genomics 2014 Dec 9;289(6):1307-19. Epub 2014 Aug 9.

Wageningen UR Plant Breeding, Wageningen University and Research Centre, 6708 PB, Wageningen, The Netherlands,

One of the most powerful technologies in unraveling the organization of a eukaryotic plant genome is high-resolution Fluorescent in situ hybridization of repeats and single copy DNA sequences on pachytene chromosomes. This technology allows the integration of physical mapping information with chromosomal positions, including centromeres, telomeres, nucleolar-organizing region, and euchromatin and heterochromatin. In this report, we established chromosomal positions of different repeat fractions of the potato genomic DNA (Cot100, Cot500 and Cot1000) on the chromosomes. We also analysed various repeat elements that are unique to potato including the moderately repetitive P5 and REP2 elements, where the REP2 is part of a larger Gypsy-type LTR retrotransposon and cover most chromosome regions, with some brighter fluorescing spots in the heterochromatin. The most abundant tandem repeat is the potato genomic repeat 1 that covers subtelomeric regions of most chromosome arms. Extensive multiple alignments of these repetitive sequences in the assembled RH89-039-16 potato BACs and the draft assembly of the DM1-3 516 R44 genome shed light on the conservation of these repeats within the potato genome. The consensus sequences thus obtained revealed the native complete transposable elements from which they were derived.
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http://dx.doi.org/10.1007/s00438-014-0891-8DOI Listing
December 2014

Fine mapping of the tomato yellow leaf curl virus resistance gene - on chromosome 11 of tomato.

Mol Breed 2014 28;34:749-760. Epub 2014 Mar 28.

Institute of Vegetable and Flowers, The Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie, Haidian District, Beijing, 100081 China.

Resistances to begomoviruses, including bipartite tomato mottle virus and monopartite tomato yellow leaf curl virus (TYLCV), have been introgressed to cultivated tomato () from wild tomato accessions. A major gene, - from f. accession "B6013 that confers resistance to TYLCV was previously mapped to a 19-cM region on the long arm of chromosome 11. In the present study, approximately 11,000 plants were screened and nearly 157 recombination events were identified between the flanking markers C2_At1g07960 (82.5 cM, physical distance 51.387 Mb) and T0302 (89 cM, 51.878 Mb). Molecular marker analysis of recombinants and TYLCV evaluation of progeny from these recombinants localized - to an approximately 300,000-bp interval between markers UP8 (51.344 Mb) and M1 (51.645 Mb). No recombinants were identified between TG36 and C2_At3g52090, a region of at least 115 kb, indicating severe recombination suppression in this region. Due to the small interval, fluorescence in situ hybridization analysis failed to clarify whether recombination suppression is caused by chromosomal rearrangements. Candidate genes predicted based on tomato genome annotation were analyzed by RT-PCR and virus-induced gene silencing. Results indicate that the NBS gene family present in the - region is likely not responsible for the --conferred resistance and that two candidate genes might play a role in the --conferred resistance. Several markers very tightly linked to the - locus are presented and useful for marker-assisted selection in breeding programs to introgress - for begomovirus resistance.
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http://dx.doi.org/10.1007/s11032-014-0072-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092234PMC
March 2014

KLK3, PCA3, and TMPRSS2-ERG expression in the peripheral blood mononuclear cell fraction from castration-resistant prostate cancer patients and response to docetaxel treatment.

Prostate 2014 Sep 7;74(12):1222-30. Epub 2014 Jul 7.

Department of Urology, Radboud University Medical Center, Nijmegen, the Netherlands.

Background: To monitor systemic disease activity, the potential of circulating tumor cells (CTCs) bears great promise. As surrogate for CTCs we measured KLK3, PCA3, and TMPRSS2-ERG messenger RNA (mRNA) in the peripheral blood mononuclear cell (PBMC) fraction from a castration-resistant prostate cancer (CRPC) patient cohort and three control groups. Moreover, biomarker response to docetaxel treatment was evaluated in the patient group.

Methods: Blood samples from 20 CRPC patients were analyzed at four different time points (prior to docetaxel treatment, at 9 weeks, 27 weeks, and 2 months after treatment). Blood was drawn once from three control groups (10 age-matched men, 10 men under 35 years of age, 12 women). All samples were analyzed for KLK3, PCA3, and TMPRSS2-ERG mRNA by using a quantitative nucleic acid amplification assay with gene-specific primers in the complementary DNA synthesis.

Results: At baseline, mRNA for KLK3 was detected in 17 (89%, 95% CI 76-100%), PCA3 in 10 (53%, 95% CI 30-75%), and TMPRSS2-ERG in seven of 19 evaluable patients (37%, 95% CI 15-59%). In contrast, the blood samples from all 32 healthy volunteers were reproducible negative for all markers. In response to docetaxel treatment, KLK3 levels decreased in 80% (95% CI 60-100%), PCA3 in 89% (95% CI 68-100%), and TMPRSS2-ERG in 86% (95% CI 60-100%) of patients.

Conclusions: The feasibility of a highly sensitive modified nucleic acid amplification assay to assess KLK3, PCA3, and TMPRSS2-ERG mRNA in the PBMC fraction from CRPC patients was demonstrated. Moreover, response of these markers to systemic treatment was shown.
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http://dx.doi.org/10.1002/pros.22839DOI Listing
September 2014

Exploring genetic variation in the tomato (Solanum section Lycopersicon) clade by whole-genome sequencing.

Plant J 2014 Oct 3;80(1):136-48. Epub 2014 Sep 3.

We explored genetic variation by sequencing a selection of 84 tomato accessions and related wild species representative of the Lycopersicon, Arcanum, Eriopersicon and Neolycopersicon groups, which has yielded a huge amount of precious data on sequence diversity in the tomato clade. Three new reference genomes were reconstructed to support our comparative genome analyses. Comparative sequence alignment revealed group-, species- and accession-specific polymorphisms, explaining characteristic fruit traits and growth habits in the various cultivars. Using gene models from the annotated Heinz 1706 reference genome, we observed differences in the ratio between non-synonymous and synonymous SNPs (dN/dS) in fruit diversification and plant growth genes compared to a random set of genes, indicating positive selection and differences in selection pressure between crop accessions and wild species. In wild species, the number of single-nucleotide polymorphisms (SNPs) exceeds 10 million, i.e. 20-fold higher than found in most of the crop accessions, indicating dramatic genetic erosion of crop and heirloom tomatoes. In addition, the highest levels of heterozygosity were found for allogamous self-incompatible wild species, while facultative and autogamous self-compatible species display a lower heterozygosity level. Using whole-genome SNP information for maximum-likelihood analysis, we achieved complete tree resolution, whereas maximum-likelihood trees based on SNPs from ten fruit and growth genes show incomplete resolution for the crop accessions, partly due to the effect of heterozygous SNPs. Finally, results suggest that phylogenetic relationships are correlated with habitat, indicating the occurrence of geographical races within these groups, which is of practical importance for Solanum genome evolution studies.
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http://dx.doi.org/10.1111/tpj.12616DOI Listing
October 2014

Fluorescence in situ hybridization and optical mapping to correct scaffold arrangement in the tomato genome.

G3 (Bethesda) 2014 May 30;4(8):1395-405. Epub 2014 May 30.

Department of Biology, Colorado State University, Fort Collins, Colorado 80523

The order and orientation (arrangement) of all 91 sequenced scaffolds in the 12 pseudomolecules of the recently published tomato (Solanum lycopersicum, 2n = 2x = 24) genome sequence were positioned based on marker order in a high-density linkage map. Here, we report the arrangement of these scaffolds determined by two independent physical methods, bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) and optical mapping. By localizing BACs at the ends of scaffolds to spreads of tomato synaptonemal complexes (pachytene chromosomes), we showed that 45 scaffolds, representing one-third of the tomato genome, were arranged differently than predicted by the linkage map. These scaffolds occur mostly in pericentric heterochromatin where 77% of the tomato genome is located and where linkage mapping is less accurate due to reduced crossing over. Although useful for only part of the genome, optical mapping results were in complete agreement with scaffold arrangement by FISH but often disagreed with scaffold arrangement based on the linkage map. The scaffold arrangement based on FISH and optical mapping changes the positions of hundreds of markers in the linkage map, especially in heterochromatin. These results suggest that similar errors exist in pseudomolecules from other large genomes that have been assembled using only linkage maps to predict scaffold arrangement, and these errors can be corrected using FISH and/or optical mapping. Of note, BAC-FISH also permits estimates of the sizes of gaps between scaffolds, and unanchored BACs are often visualized by FISH in gaps between scaffolds and thus represent starting points for filling these gaps.
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http://dx.doi.org/10.1534/g3.114.011197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132171PMC
May 2014

Hybrid recreation by reverse breeding in Arabidopsis thaliana.

Nat Protoc 2014 Apr 6;9(4):761-72. Epub 2014 Mar 6.

Laboratory of Genetics, Wageningen University, Wageningen, The Netherlands.

Hybrid crop varieties are traditionally produced by selecting and crossing parental lines to evaluate hybrid performance. Reverse breeding allows doing the opposite: selecting uncharacterized heterozygotes and generating parental lines from them. With these, the selected heterozygotes can be recreated as F1 hybrids, greatly increasing the number of hybrids that can be screened in breeding programs. Key to reverse breeding is the suppression of meiotic crossovers in a hybrid plant to ensure the transmission of nonrecombinant chromosomes to haploid gametes. These gametes are subsequently regenerated as doubled-haploid (DH) offspring. Each DH carries combinations of its parental chromosomes, and complementing pairs can be crossed to reconstitute the initial hybrid. Achiasmatic meiosis and haploid generation result in uncommon phenotypes among offspring owing to chromosome number variation. We describe how these features can be dealt with during a reverse-breeding experiment, which can be completed in six generations (∼1 year).
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http://dx.doi.org/10.1038/nprot.2014.049DOI Listing
April 2014

The genomic landscape of meiotic crossovers and gene conversions in Arabidopsis thaliana.

Elife 2013 Dec 17;2:e01426. Epub 2013 Dec 17.

Laboratory of Genetics, Wageningen University, Wageningen, Netherlands.

Knowledge of the exact distribution of meiotic crossovers (COs) and gene conversions (GCs) is essential for understanding many aspects of population genetics and evolution, from haplotype structure and long-distance genetic linkage to the generation of new allelic variants of genes. To this end, we resequenced the four products of 13 meiotic tetrads along with 10 doubled haploids derived from Arabidopsis thaliana hybrids. GC detection through short reads has previously been confounded by genomic rearrangements. Rigid filtering for misaligned reads allowed GC identification at high accuracy and revealed an ∼80-kb transposition, which undergoes copy-number changes mediated by meiotic recombination. Non-crossover associated GCs were extremely rare most likely due to their short average length of ∼25-50 bp, which is significantly shorter than the length of CO-associated GCs. Overall, recombination preferentially targeted non-methylated nucleosome-free regions at gene promoters, which showed significant enrichment of two sequence motifs. DOI: http://dx.doi.org/10.7554/eLife.01426.001.
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http://dx.doi.org/10.7554/eLife.01426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3865688PMC
December 2013

Fluorescent In Situ Hybridization (FISH) on pachytene chromosomes as a tool for genome characterization.

Methods Mol Biol 2013 ;1069:15-24

Laboratory of Molecular Biology, Wageningen University, Wageningen, The Netherlands.

A growing number of international genome consortia have initiated large-scale sequencing projects for most of the major crop species. This huge amount of information not only boosted genetic and physical mapping research, but it also enabled novel applications on the level of chromosome biology including molecular cytogenetics that supports plant genetics, genomics, and breeding programs. The simultaneous detection of a large number of BAC-based probes by multicolor fluorescent in situ hybridization (FISH) can provide a rapid overview of super-contig and gap distribution on euchromatin chromosome areas and will display directly and precisely the positions of chromosome rearrangements. Furthermore, hybridizations of BACs on the chromosomes of related species can confirm genomic colinearity, or the occurrence of inversions and translocations events. This cross-species FISH together with meiotic pairing studies is a powerful source of information that elucidates the nature of genome rearrangements, and the consequences of such rearrangements for introgressive hybridizations. In this chapter we describe a general-purpose protocol for FISH on pachytene chromosomes.
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http://dx.doi.org/10.1007/978-1-62703-613-9_2DOI Listing
March 2014

Prospective multicentre evaluation of PCA3 and TMPRSS2-ERG gene fusions as diagnostic and prognostic urinary biomarkers for prostate cancer.

Eur Urol 2014 Mar 15;65(3):534-42. Epub 2012 Nov 15.

Radboud University Nijmegen Medical Centre, Department of Urology, Nijmegen, The Netherlands. Electronic address:

Background: Prostate cancer antigen 3 (PCA3) and v-ets erythroblastosis virus E26 oncogene homolog (TMPRSS2-ERG) gene fusions are promising prostate cancer (PCa) specific biomarkers that can be measured in urine.

Objective: To evaluate the diagnostic and prognostic value of Progensa PCA3 and TMPRSS2-ERG gene fusions (as individual biomarkers and as a panel) for PCa in a prospective multicentre setting.

Design, Setting, And Participants: At six centres, post-digital rectal examination first-catch urine specimens prior to prostate biopsies were prospectively collected from 497 men. We assessed the predictive value of Progensa PCA3 and TMPRSS2-ERG (quantitative nucleic acid amplification assay to detect TMPRSS2-ERG messenger RNA [mRNA]) for PCa, Gleason score, clinical tumour stage, and PCa significance (individually and as a marker panel). This was compared with serum prostate-specific antigen and the European Randomised Study of Screening for Prostate Cancer (ERSPC) risk calculator. In a subgroup (n=61) we evaluated biomarker association with prostatectomy outcome.

Outcome Measurements And Statistical Analysis: Univariate and multivariate logistic regression analysis and receiver operating curves were used.

Results And Limitations: Urine samples of 443 men contained sufficient mRNA for marker analysis. PCa was diagnosed in 196 of 443 men. Both PCA3 and TMPRSS2-ERG had significant additional predictive value to the ERSPC risk calculator parameters in multivariate analysis (p<0.001 and resp. p=0.002). The area under the curve (AUC) increased from 0.799 (ERSPC risk calculator), to 0.833 (ERSPC risk calculator plus PCA3), to 0.842 (ERSPC risk calculator plus PCA3 plus TMPRSS2-ERG) to predict PCa. Sensitivity of PCA3 increased from 68% to 76% when combined with TMPRSS2-ERG. TMPRSS2-ERG added significant predictive value to the ERSPC risk calculator to predict biopsy Gleason score (p<0.001) and clinical tumour stage (p=0.023), whereas PCA3 did not.

Conclusions: TMPRSS2-ERG had independent additional predictive value to PCA3 and the ERSPC risk calculator parameters for predicting PCa. TMPRSS2-ERG had prognostic value, whereas PCA3 did not. Implementing the novel urinary biomarker panel PCA3 and TMPRSS2-ERG into clinical practice would lead to a considerable reduction of the number of prostate biopsies.
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http://dx.doi.org/10.1016/j.eururo.2012.11.014DOI Listing
March 2014

Chromosome evolution in Solanum traced by cross-species BAC-FISH.

New Phytol 2012 Aug 11;195(3):688-698. Epub 2012 Jun 11.

Wageningen UR Plant Breeding, Droevendaalsesteeg 1, 6708 PB Wageningen, the Netherlands.

Chromosomal rearrangements are relatively rare evolutionary events and can be used as markers to study karyotype evolution. This research aims to use such rearrangements to study chromosome evolution in Solanum. Chromosomal rearrangements between Solanum crops and several related wild species were investigated using tomato and potato bacterial artificial chromosomes (BACs) in a multicolour fluorescent in situ hybridization (FISH). The BACs selected are evenly distributed over seven chromosomal arms containing inversions described in previous studies. The presence/absence of these inversions among the studied Solanum species were determined and the order of the BAC-FISH signals was used to construct phylogenetic trees.Compared with earlier studies, data from this study provide support for the current grouping of species into different sections within Solanum; however, there are a few notable exceptions, such as the tree positions of S. etuberosum (closer to the tomato group than to the potato group) and S. lycopersicoides (sister to S. pennellii). These apparent contradictions might be explained by interspecific hybridization events and/or incomplete lineage sorting. This cross-species BAC painting technique provides unique information on genome organization, evolution and phylogenetic relationships in a wide variety of species. Such information is very helpful for introgressive breeding.
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http://dx.doi.org/10.1111/j.1469-8137.2012.04195.xDOI Listing
August 2012

Structural homology in the Solanaceae: analysis of genomic regions in support of synteny studies in tomato, potato and pepper.

Plant J 2012 Aug 12;71(4):602-14. Epub 2012 Jun 12.

Plant Research International, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.

We have analysed the structural homology in euchromatin regions of tomato, potato and pepper with special attention for the long arm of chromosome 2 (2L). Molecular organization and colinear junctions were delineated using multi-color BAC FISH analysis and comparative sequence alignment. We found large-scale rearrangements including inversions and segmental translocations that were not reported in previous comparative studies. Some of the structural rearrangements are specific for the tomato clade, and differentiate tomato from potato, pepper and other Solanaceous species. Although local gene vicinity is largely preserved, there are many small-scale synteny perturbations. Gene adjacency in the aligned segments was frequently disrupted for 47% of the ortholog pairs as a result of gene and LTR retrotransposon insertions, and occasionally by single gene inversions and translocations. Our data also suggests that long distance intra-chromosomal rearrangements and local gene rearrangements have evolved frequently during speciation in the Solanum genus, and that small changes are more prevalent than large-scale differences. The occurrence of sonata and harbinger transposable elements and other repeats near or at junction breaks is considered in the light of repeat-mediated rearrangements and a reconstruction scenario for an ancestral 2L topology is discussed.
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http://dx.doi.org/10.1111/j.1365-313X.2012.05012.xDOI Listing
August 2012

Reverse breeding in Arabidopsis thaliana generates homozygous parental lines from a heterozygous plant.

Nat Genet 2012 Mar 11;44(4):467-70. Epub 2012 Mar 11.

Laboratory of Genetics, Wageningen University, Wageningen, The Netherlands.

Traditionally, hybrid seeds are produced by crossing selected inbred lines. Here we provide a proof of concept for reverse breeding, a new approach that simplifies meiosis such that homozygous parental lines can be generated from a vigorous hybrid individual. We silenced DMC1, which encodes the meiotic recombination protein DISRUPTED MEIOTIC cDNA1, in hybrids of A. thaliana, so that non-recombined parental chromosomes segregate during meiosis. We then converted the resulting gametes into adult haploid plants, and subsequently into homozygous diploids, so that each contained half the genome of the original hybrid. From 36 homozygous lines, we selected 3 (out of 6) complementing parental pairs that allowed us to recreate the original hybrid by intercrossing. In addition, this approach resulted in a complete set of chromosome-substitution lines. Our method allows the selection of a single choice offspring from a segregating population and preservation of its heterozygous genotype by generating homozygous founder lines.
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http://dx.doi.org/10.1038/ng.2203DOI Listing
March 2012