Insights into Sex Chromosome Evolution and Aging from the Genome of a Short-Lived Fish.

The killifish Nothobranchius furzeri is the shortest-lived vertebrate that can be bred in the laboratory. Its rapid growth, early sexual maturation, fast aging, and arrested embryonic development (diapause) make it an attractive model organism in biomedical research. Here, we report a draft sequence of its genome that allowed us to uncover an intra-species Y chromosome polymorphism representing-in real time-different stages of sex chromosome formation that display features of early mammalian XY evolution "in action." Our data suggest that gdf6Y, encoding a TGF-ß family growth factor, is the master sex-determining gene in N. furzeri. Moreover, we observed genomic clustering of aging-related genes, identified genes under positive selection, and revealed significant similarities of gene expression profiles between diapause and aging, particularly for genes controlling cell cycle and translation. The annotated genome sequence is provided as an online resource (http://www.nothobranchius.info/NFINgb).

Effects of ligandrol as a selective androgen receptor modulator in a rat model for osteoporosis.

INTRODUCTION: The selective androgen receptor modulator ligandrol (LGD-4033 or VK5211) has been shown to improve muscle tissue. In the present study, the effect of ligandrol on bone tissue was investigated in ovariectomized rat model. MATERIALS AND METHODS: Three-month-old Sprague Dawley rats were either ovariectomized (OVX, n = 60) or left intact (NON-OVX, n = 15). After 9 weeks, OVX rats were divided into four groups: untreated OVX (n = 15) group and three OVX groups (each of 15 rats) treated with ligandrol orally at doses of 0.03, 0.3, or 3 mg/kg body weight. After five weeks, lumbar vertebral bodies (L), tibiae, and femora were examined using micro-computed tomographical, biomechanical, ashing, and gene expression analyses. RESULTS: In the 3-mg ligandrol group, bone structural properties were improved (trabecular number: 38 ± 8 vs. 35 ± 7 (femur), 26 ± 7 vs. 22 ± 6 (L), 12 ± 5 vs. 6 ± 3 (tibia) and serum phosphorus levels (1.81 ± 0.17 vs.1.41 ± 0.17 mmol/l), uterus (0.43 ± 0.04 vs. 0.11 ± 0.02 g), and heart (1.13 ± 0.11 vs. 1.01 ± 0.08 g) weights were increased compared to the OVX group. Biomechanical parameters were not changed. Low and medium doses did not affect bone tissue and had fewer side effects. Body weight and food intake were not affected by ligandrol; OVX led to an increase in these parameters and worsened all bone parameters. CONCLUSION: Ligandrol at high dose showed a subtle anabolic effect on structural properties without any improvement in biomechanical properties of osteoporotic bones. Considering side effects of ligandrol at this dose, its further investigation for the therapy of postmenopausal osteoporosis should be reevaluated.

A chromosome conformation capture ordered sequence of the barley genome.

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

A Homolog of Blade-On-Petiole 1 and 2 (BOP1/2) Controls Internode Length and Homeotic Changes of the Barley Inflorescence.

Inflorescence architecture in small-grain cereals has a direct effect on yield and is an important selection target in breeding for yield improvement. We analyzed the recessive mutation laxatum-a (lax-a) in barley (Hordeum vulgare), which causes pleiotropic changes in spike development, resulting in (1) extended rachis internodes conferring a more relaxed inflorescence, (2) broadened base of the lemma awns, (3) thinner grains that are largely exposed due to reduced marginal growth of the palea and lemma, and (4) and homeotic conversion of lodicules into two stamenoid structures. Map-based cloning enforced by mapping-by-sequencing of the mutant lax-a locus enabled the identification of a homolog of BLADE-ON-PETIOLE1 (BOP1) and BOP2 as the causal gene. Interestingly, the recently identified barley uniculme4 gene also is a BOP1/2 homolog and has been shown to regulate tillering and leaf sheath development. While the Arabidopsis (Arabidopsis thaliana) BOP1 and BOP2 genes act redundantly, the barley genes contribute independent effects in specifying the developmental growth of vegetative and reproductive organs, respectively. Analysis of natural genetic diversity revealed strikingly different haplotype diversity for the two paralogous barley genes, likely affected by the respective genomic environments, since no indication for an active selection process was detected.

The distribution of α-kleisin during meiosis in the holocentromeric plant Luzula elegans.

Holocentric chromosomes occur in a number of independent eukaryotic lineages, and they form holokinetic kinetochores along the entire poleward chromatid surfaces. Due to this alternative chromosome structure, Luzula elegans sister chromatids segregate already in anaphase I followed by the segregation of the homologues in anaphase II. However, not yet known is the localization and dynamics of cohesin and the structure of the synaptonemal complex (SC) during meiosis. We show here that the α-kleisin subunit of cohesin localizes at the centromeres of both mitotic and meiotic metaphase chromosomes and that it, thus, may contribute to assemble the centromere in L. elegans. This localization and the formation of a tripartite SC structure indicate that the prophase I behaviour of L. elegans is similar as in monocentric species.

Comparative genomics to explore phylogenetic relationship, cryptic sexual potential and host specificity of Rhynchosporium species on grasses.

BACKGROUND: The Rhynchosporium species complex consists of hemibiotrophic fungal pathogens specialized to different sweet grass species including the cereal crops barley and rye. A sexual stage has not been described, but several lines of evidence suggest the occurrence of sexual reproduction. Therefore, a comparative genomics approach was carried out to disclose the evolutionary relationship of the species and to identify genes demonstrating the potential for a sexual cycle. Furthermore, due to the evolutionary very young age of the five species currently known, this genus appears to be well-suited to address the question at the molecular level of how pathogenic fungi adapt to their hosts. RESULTS: The genomes of the different Rhynchosporium species were sequenced, assembled and annotated using ab initio gene predictors trained on several fungal genomes as well as on Rhynchosporium expressed sequence tags. Structures of the rDNA regions and genome-wide single nucleotide polymorphisms provided a hypothesis for intra-genus evolution. Homology screening detected core meiotic genes along with most genes crucial for sexual recombination in ascomycete fungi. In addition, a large number of cell wall-degrading enzymes that is characteristic for hemibiotrophic and necrotrophic fungi infecting monocotyledonous hosts were found. Furthermore, the Rhynchosporium genomes carry a repertoire of genes coding for polyketide synthases and non-ribosomal peptide synthetases. Several of these genes are missing from the genome of the closest sequenced relative, the poplar pathogen Marssonina brunnea, and are possibly involved in adaptation to the grass hosts. Most importantly, six species-specific genes coding for protein effectors were identified in R. commune. Their deletion yielded mutants that grew more vigorously in planta than the wild type. CONCLUSION: Both cryptic sexuality and secondary metabolites may have contributed to host adaptation. Most importantly, however, the growth-retarding activity of the species-specific effectors suggests that host adaptation of R. commune aims at extending the biotrophic stage at the expense of the necrotrophic stage of pathogenesis. Like other apoplastic fungi Rhynchosporium colonizes the intercellular matrix of host leaves relatively slowly without causing symptoms, reminiscent of the development of endophytic fungi. Rhynchosporium may therefore become an object for studying the mutualism-parasitism transition.

Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes.

Hierarchical shotgun sequencing remains the method of choice for assembling high-quality reference sequences of complex plant genomes. The efficient exploitation of current high-throughput technologies and powerful computational facilities for large-insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole-genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high-quality assemblies of a large number of clones to assemble map-based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path.

A sequence-ready physical map of barley anchored genetically by two million single-nucleotide polymorphisms.

Barley (Hordeum vulgare) is an important cereal crop and a model species for Triticeae genomics. To lay the foundation for hierarchical map-based sequencing, a genome-wide physical map of its large and complex 5.1 billion-bp genome was constructed by high-information content fingerprinting of almost 600,000 bacterial artificial chromosomes representing 14-fold haploid genome coverage. The resultant physical map comprises 9,265 contigs with a cumulative size of 4.9 Gb representing 96% of the physical length of the barley genome. The reliability of the map was verified through extensive genetic marker information and the analysis of topological networks of clone overlaps. A minimum tiling path of 66,772 minimally overlapping clones was defined that will serve as a template for hierarchical clone-by-clone map-based shotgun sequencing. We integrated whole-genome shotgun sequence data from the individuals of two mapping populations with published bacterial artificial chromosome survey sequence information to genetically anchor the physical map. This novel approach in combination with the comprehensive whole-genome shotgun sequence data sets allowed us to independently validate and improve a previously reported physical and genetic framework. The resources developed in this study will underpin fine-mapping and cloning of agronomically important genes and the assembly of a draft genome sequence.

The holocentric species Luzula elegans shows interplay between centromere and large-scale genome organization.

In higher plants, the large-scale structure of monocentric chromosomes consists of distinguishable eu- and heterochromatic regions, the proportions and organization of which depend on a species' genome size. To determine whether the same interplay is maintained for holocentric chromosomes, we investigated the distribution of repetitive sequences and epigenetic marks in the woodrush Luzula elegans (3.81 Gbp/1C). Sixty-one per cent of the L. elegans genome is characterized by highly repetitive DNA, with over 30 distinct sequence families encoding an exceptionally high diversity of satellite repeats. Over 33% of the genome is composed of the Angela clade of Ty1/copia LTR retrotransposons, which are uniformly dispersed along the chromosomes, while the satellite repeats occur as bands whose distribution appears to be biased towards the chromosome termini. No satellite showed an almost chromosome-wide distribution pattern as expected for a holocentric chromosome and no typical centromere-associated LTR retrotransposons were found either. No distinguishable large-scale patterns of eu- and heterochromatin-typical epigenetic marks or early/late DNA replicating domains were found along mitotic chromosomes, although super-high-resolution light microscopy revealed distinguishable interspersed units of various chromatin types. Our data suggest a correlation between the centromere and overall genome organization in species with holocentric chromosomes.

8p23 beta-defensin copy number determination by single-locus pseudogene-based paralog ratio tests risk bias due to low-frequency sequence variations.

BACKGROUND: The copy number variation (CNV) in beta-defensin genes (DEFB) on human chromosome 8p23 has been proposed to contribute to the phenotypic differences in inflammatory diseases. However, determination of exact DEFB CN is a major challenge in association studies. Quantitative real-time PCR (qPCR), paralog ratio tests (PRT) and multiplex ligation-dependent probe amplification (MLPA) have been extensively used to determine DEFB CN in different laboratories, but inter-method inconsistencies were observed frequently. In this study we asked which one is superior among the three methods for DEFB CN determination. RESULTS: We developed a clustering approach for MLPA and PRT to statistically correlate data from a single experiment. Then we compared qPCR, a newly designed PRT and MLPA for DEFB CN determination in 285 DNA samples. We found MLPA had the best convergence and clustering results of the raw data and the highest call rate. In addition, the concordance rates between MLPA or PRT and qPCR (32.12% and 37.99%, respectively) were unacceptably low with underestimated CN by qPCR. Concordance rate between MLPA and PRT (90.52%) was high but PRT systematically underestimated CN by one in a subset of samples. In these samples a sequence variant which caused complete PCR dropout of the respective DEFB cluster copies was found in one primer binding site of one of the targeted paralogous pseudogenes. CONCLUSION: MLPA is superior to PRT and even more to qPCR for DEFB CN determination. Although the applied PRT provides in most cases reliable results, such a test is particularly sensitive to low-frequency sequence variations preferably accumulating in loci like pseudogenes which are most likely not under selective pressure. In the light of the superior performance of multiplex assays, the drawbacks of such single PRTs could be overcome by combining more test markers.

Comparative analysis of mitochondrial genomes from closely related Rhynchosporium species reveals extensive intron invasion.

We sequenced and annotated the complete mitochondrial (mt) genomes of four closely related Rhynchosporium species that diverged ∼14,000-35,000years ago. During this time frame, three of the mt genomes expanded significantly due to an invasion of introns into three genes (cox1, cox2, and nad5). The enlarged mt genomes contained ∼40% introns compared to 8.1% in uninvaded relatives. Many intron gains were accompanied by co-conversion of flanking exonic regions. The comparative analysis revealed a highly variable set of non-intronic, free-standing ORFs of unknown function (uORFs). This is consistent with a rapidly evolving accessory compartment in the mt genome of these closely related species. Only one free-standing uORF was shared among all mt genomes analyzed. This uORF had a mutation rate similar to the core mt protein-encoding genes, suggesting conservation of function among the species. The nucleotide composition of the core protein-encoding genes significantly differed from those of introns and uORFs. The mt mutation rate was 77 times higher than the nuclear mutation rate, indicating that the phylogeny inferred from mt genes may better resolve the phylogenetic relationships among closely related Rhynchosporium species than phylogenies inferred from nuclear genes.

Frequent gene movement and pseudogene evolution is common to the large and complex genomes of wheat, barley, and their relatives.

All six arms of the group 1 chromosomes of hexaploid wheat (Triticum aestivum) were sequenced with Roche/454 to 1.3- to 2.2-fold coverage and compared with similar data sets from the homoeologous chromosome 1H of barley (Hordeum vulgare). Six to ten thousand gene sequences were sampled per chromosome. These were classified into genes that have their closest homologs in the Triticeae group 1 syntenic region in Brachypodium, rice (Oryza sativa), and/or sorghum (Sorghum bicolor) and genes that have their homologs elsewhere in these model grass genomes. Although the number of syntenic genes was similar between the homologous groups, the amount of nonsyntenic genes was found to be extremely diverse between wheat and barley and even between wheat subgenomes. Besides a small core group of genes that are nonsyntenic in other grasses but conserved among Triticeae, we found thousands of genic sequences that are specific to chromosomes of one single species or subgenome. By examining in detail 50 genes from chromosome 1H for which BAC sequences were available, we found that many represent pseudogenes that resulted from transposable element activity and double-strand break repair. Thus, Triticeae seem to accumulate nonsyntenic genes frequently. Since many of them are likely to be pseudogenes, total gene numbers in Triticeae are prone to pronounced overestimates.

Unlocking the barley genome by chromosomal and comparative genomics.

We used a novel approach that incorporated chromosome sorting, next-generation sequencing, array hybridization, and systematic exploitation of conserved synteny with model grasses to assign ~86% of the estimated ~32,000 barley (Hordeum vulgare) genes to individual chromosome arms. Using a series of bioinformatically constructed genome zippers that integrate gene indices of rice (Oryza sativa), sorghum (Sorghum bicolor), and Brachypodium distachyon in a conserved synteny model, we were able to assemble 21,766 barley genes in a putative linear order. We show that the barley (H) genome displays a mosaic of structural similarity to hexaploid bread wheat (Triticum aestivum) A, B, and D subgenomes and that orthologous genes in different grasses exhibit signatures of positive selection in different lineages. We present an ordered, information-rich scaffold of the barley genome that provides a valuable and robust framework for the development of novel strategies in cereal breeding.

The transcript catalogue of the short-lived fish Nothobranchius furzeri provides insights into age-dependent changes of mRNA levels.

BACKGROUND: The African annual fish Nothobranchius furzeri has over recent years been established as a model species for ageing-related studies. This is mainly based on its exceptionally short lifespan and the presence of typical characteristics of vertebrate ageing. To substantiate its role as an alternative vertebrate ageing model, a transcript catalogue is needed, which can serve e.g. as basis for identifying ageing-related genes. RESULTS: To build the N. furzeri transcript catalogue, thirteen cDNA libraries were sequenced using Sanger, 454/Roche and Solexa/Illumina technologies yielding about 39 Gb. In total, 19,875 protein-coding genes were identified and annotated. Of these, 71% are represented by at least one transcript contig with a complete coding sequence. Further, transcript levels of young and old fish of the strains GRZ and MZM-0403, which differ in lifespan by twofold, were studied by RNA-seq. In skin and brain, 85 differentially expressed genes were detected; these have a role in cell cycle control and proliferation, inflammation and tissue maintenance. An RNA-seq experiment for zebrafish skin confirmed the ageing-related relevance of the findings in N. furzeri. Notably, analyses of transcript levels between zebrafish and N. furzeri but also between N. furzeri strains differed largely, suggesting that ageing is accelerated in the short-lived N. furzeri strain GRZ compared to the longer-lived strain MZM-0403. CONCLUSIONS: We provide a comprehensive, annotated N. furzeri transcript catalogue and a first transcriptome-wide insight into N. furzeri ageing. This data will serve as a basis for future functional studies of ageing-related genes.

BSTA: a targeted approach combines bulked segregant analysis with next- generation sequencing and de novo transcriptome assembly for SNP discovery in sunflower.

BACKGROUND: Sunflower belongs to the largest plant family on earth, the genomically poorly explored Compositae. Downy mildew Plasmopara halstedii (Farlow) Berlese & de Toni is one of the major diseases of cultivated sunflower (Helianthus annuus L.). In the search for new sources of downy mildew resistance, the locus Pl(ARG)on linkage group 1 (LG1) originating from H. argophyllus is promising since it confers resistance against all known races of the pathogen. However, the mapping resolution in the Pl(ARG) region is hampered by significantly suppressed recombination and by limited availability of polymorphic markers. Here we examined a strategy developed for the enrichment of molecular markers linked to this specific genomic region. We combined bulked segregant analysis (BSA) with next-generation sequencing (NGS) and de novo assembly of the sunflower transcriptome for single nucleotide polymorphism (SNP) discovery in a sequence resource combining reads originating from two sunflower species, H. annuus and H. argophyllus. RESULTS: A computational pipeline developed for SNP calling and pattern detection identified 219 candidate genes. For a proof of concept, 42 resistance gene-like sequences were subjected to experimental SNP validation. Using a high-resolution mapping population, 12 SNP markers were mapped to LG1. We successfully verified candidate sequences either co-segregating with or closely flanking Pl(ARG). CONCLUSIONS: This study is the first successful example to improve bulked segregant analysis with de novo transcriptome assembly using next generation sequencing. The BSTA pipeline we developed provides a useful guide for similar studies in other non-model organisms. Our results demonstrate this method is an efficient way to enrich molecular markers and to identify candidate genes in a specific mapping interval.

Sequencing of 21 varicella-zoster virus genomes reveals two novel genotypes and evidence of recombination.

Genotyping of 21 varicella-zoster virus (VZV) strains using a scattered single nucleotide polymorphism (SNP) method revealed ambiguous SNPs and two nontypeable isolates. For a further genetic characterization, the genomes of all strains were sequenced using the 454 technology. Almost-complete genome sequences were assembled, and most remaining gaps were closed with Sanger sequencing. Phylogenetic analysis of 42 genomes revealed five established and two novel VZV genotypes, provisionally termed VIII and IX. Genotypes VIII and IX are distinct from the previously reported provisional genotypes VI and VII as judged from the SNP pattern. The alignments showed evidence of ancient recombination events in the phylogeny of clade 4 and recent recombinations within single strains: 3/2005 (clade 1), 11 and 405/2007 (clade 3), 8 and DR (clade 4), CA123 and 413/2000 (clade 5), and strains of the novel genotypes VIII and IX. Bayesian tree inference of the thymidine kinase and the polymerase genes of the VZV clades and other varicelloviruses revealed that VZV radiation began some 110,000 years ago, which correlates with the out-of-Africa dispersal of modern humans. The split of ancestral clades 2/4 and 1/3/5/VIII/IX shows the greatest node height.

Genomics-based high-resolution mapping of the BaMMV/BaYMV resistance gene rym11 in barley (Hordeum vulgare L.).

Soil-borne barley yellow mosaic virus disease, caused by different strains of Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), is one of the most important diseases of winter barley (Hordeum vulgare L.) in Europe and East Asia. The recessive resistance gene rym11 located in the centromeric region of chromosome 4HL is effective against all so far known strains of BaMMV and BaYMV in Germany. In order to isolate this gene, a high-resolution mapping population (10,204 meiotic events) has been constructed. F2 plants were screened with co-dominant flanking markers and segmental recombinant inbred lines (RILs) were tested for resistance to BaMMV under growth chamber and field conditions. Tightly linked markers were developed by exploiting (1) publicly available barley EST sequences, (2) employing barley synteny to rice, Brachypodium distachyon and sorghum and (3) using next-generation sequencing data of barley. Using this approach, the genetic interval was efficiently narrowed down from the initial 10.72 % recombination to 0.074 % recombination. A marker co-segregating with rym11 was developed providing the basis for gene isolation and efficient marker-assisted selection.

Screening for Resistant Bacteria, Antimicrobial Resistance Genes, Sexually Transmitted Infections and Schistosoma spp. in Tissue Samples from Predominantly Vaginally Delivered Placentae in Ivory Coast and Ghana.

Medical complications during pregnancy have been frequently reported from Western Africa with a particular importance of infectious complications. Placental tissue can either become the target of infectious agents itself, such as, e.g., in the case of urogenital schistosomiasis, or be subjected to contamination with colonizing or infection-associated microorganisms of the cervix or the vagina during vaginal delivery. In the retrospective cross-sectional assessment presented here, the quantitative dimension of infection or colonization with selected resistant or pathogenic bacteria and parasites was regionally assessed. To do so, 274 collected placental tissues from Ivory Coastal and Ghanaian women were subjected to selective growth of resistant bacteria, as well as to molecular screening for beta-lactamase genes, Schistosoma spp. and selected bacterial causative agents of sexually transmitted infections (STI). Panton-Valentine-negative methicillin-resistant Staphylococcus aureus (MRSA) was grown from 1.8% of the tissue samples, comprising the spa types t008 and t688, as well as the newly detected ones, t12101 (n = 2) and t12102. While the culture-based recovery of resistant Enterobacterales and nonfermentative rod-shaped Gram-negative bacteria failed, molecular assessments confirmed beta-lactamase genes in 31.0% of the samples with multiple detections of up to four resistance genes per sample and blaCTX-M, blaIMP, blaGES, blaVIM, blaOXA-58-like, blaNDM, blaOXA-23-like, blaOXA-48-like and blaKPC occurring in descending order of frequency. The beta-lactamase genes blaOXA-40/24-like, blaNMC_A/IMI, blaBIC, blaSME, blaGIM and blaDIM were not detected. DNA of the urogenital schistosomiasis-associated Schistosoma haematobium complex was recorded in 18.6% of the samples, but only a single positive signal for S. mansoni with a high cycle-threshold value in real-time PCR was found. Of note, higher rates of schistosomiasis were observed in Ghana (54.9% vs. 10.3% in Ivory Coast) and Cesarean section was much more frequent in schistosomiasis patients (61.9% vs. 14.8% in women without Schistosoma spp. DNA in the placenta). Nucleic acid sequences of nonlymphogranuloma-venereum-associated Chlamydia trachomatis and of Neisseria gonorrhoeae were recorded in 1.1% and 1.9% of the samples, respectively, while molecular attempts to diagnose Treponema pallidum and Mycoplasma genitalium did not lead to positive results. Molecular detection of Schistosoma spp. or STI-associated pathogens was only exceptionally associated with multiple resistance gene detections in the same sample, suggesting epidemiological distinctness. In conclusion, the assessment confirmed considerable prevalence of urogenital schistosomiasis and resistant bacterial colonization, as well as a regionally expected abundance of STI-associated pathogens. Continuous screening offers seem advisable to minimize the risks for the pregnant women and their newborns.

A highly conserved retinoic acid responsive element controls wt1a expression in the zebrafish pronephros.

The Wilms' tumor suppressor gene Wt1 encodes a zinc-finger transcription factor that plays an essential role in organ development, most notably of the kidney. Despite its importance for organogenesis, knowledge of the regulation of Wt1 expression is scarce. Here, we have used transgenesis in zebrafish harboring two wt1 genes, wt1a and wt1b, in order to define regulatory elements that drive wt1 expression in the kidney. Stable transgenic lines with approximately 30 kb of the upstream genomic regions of wt1a or wt1b almost exactly recapitulated endogenous expression of the wt1 paralogs. In the case of wt1b, we have identified an enhancer that is located in the far upstream region that is necessary and sufficient for reporter gene expression in the pronephric glomeruli. Regarding wt1a, we could also identify an enhancer that is located approximately 4 kb upstream of the transcriptional start site that is required for expression in the intermediate mesoderm. Interestingly, this intermediate mesoderm enhancer is highly conserved between fish and mammals, is bound by members of the retinoic acid receptor family of transcription factors in gel shift experiments and mediates responsiveness to retinoic acid both in vivo and in cell culture. To our knowledge, this is the first functional demonstration of defined regulatory elements controlling Wt1 expression in vivo. The identification of kidney-specific enhancer elements will help us to better understand the integration of extracellular signals into intracellular networks in nephrogenesis.

Molecular, phylogenetic and comparative genomic analysis of the cytokinin oxidase/dehydrogenase gene family in the Poaceae.

The genomes of cereals such as wheat (Triticum aestivum) and barley (Hordeum vulgare) are large and therefore problematic for the map-based cloning of agronomicaly important traits. However, comparative approaches within the Poaceae permit transfer of molecular knowledge between species, despite their divergence from a common ancestor sixty million years ago. The finding that null variants of the rice gene cytokinin oxidase/dehydrogenase 2 (OsCKX2) result in large yield increases provides an opportunity to explore whether similar gains could be achieved in other Poaceae members. Here, phylogenetic, molecular and comparative analyses of CKX families in the sequenced grass species rice, brachypodium, sorghum, maize and foxtail millet, as well as members identified from the transcriptomes/genomes of wheat and barley, are presented. Phylogenetic analyses define four Poaceae CKX clades. Comparative analyses showed that CKX phylogenetic groupings can largely be explained by a combination of local gene duplication, and the whole-genome duplication event that predates their speciation. Full-length OsCKX2 homologues in barley (HvCKX2.1, HvCKX2.2) and wheat (TaCKX2.3, TaCKX2.4, TaCKX2.5) are characterized, with comparative analysis at the DNA, protein and genetic/physical map levels suggesting that true CKX2 orthologs have been identified. Furthermore, our analysis shows CKX2 genes in barley and wheat have undergone a Triticeae-specific gene-duplication event. Finally, by identifying ten of the eleven CKX genes predicted to be present in barley by comparative analyses, we show that next-generation sequencing approaches can efficiently determine the gene space of large-genome crops. Together, this work provides the foundation for future functional investigation of CKX family members within the Poaceae.