Frank Panitz

Expanded retina territory by midbrain transformation upon overexpression of Six6 (Optx2) in Xenopus embryos

During vertebrate eye development, the expression of the homeobox gene Six6 is restricted to the neural retina and is initiated later than Rx and Pax6 in the presumptive retina field. We show here that overexpression of mouse Six6 in Xenopus embryos can induce transformation of competent tissue of the anterior neural plate into retinal tissue. In Six6 injected embryos, the molecular identity of the presumptive midbrain and rostral hindbrain regions was lost, as shown by the absence of XEn-2 and Xpax2 expression, being replaced by the ectopic expression of the retinal markers Xpax6 and Xrx. When allowed to grow further, Six6 injected embryos developed ectopic eye-like structures in the rostral brain and showed a transformation of the midbrain into retina. Similar results were obtained upon overexpression of Six3 or Xsix3, revealing a possible redundance of Six3 and Six6 activities. Taken together, results obtained suggest that during normal retina development, the relatively late expressed Six6 gene becomes part of a network of retinal homeobox genes that are linked together by positive feedback loops. Furthermore, our results demonstrate that the primitive neural ectoderm of the future midbrain and rostral hindbrain is competent to form retinal tissue.

Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags

BackgroundKnowledge of the structure of gene expression is essential for mammalian transcriptomics research. We analyzed a collection of more than one million porcine expressed sequence tags (ESTs), of which two-thirds were generated in the Sino-Danish Pig Genome Project and one-third are from public databases. The Sino-Danish ESTs were generated from one normalized and 97 non-normalized cDNA libraries representing 35 different tissues and three developmental stages.ResultsUsing the Distiller package, the ESTs were assembled to roughly 48,000 contigs and 73,000 singletons, of which approximately 25% have a high confidence match to UniProt. Approximately 6,000 new porcine gene clusters were identified. Expression analysis based on the non-normalized libraries resulted in the following findings. The distribution of cluster sizes is scaling invariant. Brain and testes are among the tissues with the greatest number of different expressed genes, whereas tissues with more specialized function, such as developing liver, have fewer expressed genes. There are at least 65 high confidence housekeeping gene candidates and 876 cDNA library-specific gene candidates. We identified differential expression of genes between different tissues, in particular brain/spinal cord, and found patterns of correlation between genes that share expression in pairs of libraries. Finally, there was remarkable agreement in expression between specialized tissues according to Gene Ontology categories.ConclusionThis EST collection, the largest to date in pig, represents an essential resource for annotation, comparative genomics, assembly of the pig genome sequence, and further porcine transcription studies.

A 660-Kb Deletion with Antagonistic Effects on Fertility and Milk Production Segregates at High Frequency in Nordic Red Cattle: Additional Evidence for the Common Occurrence of Balancing Selection in Livestock

In dairy cattle, the widespread use of artificial insemination has resulted in increased selection intensity, which has led to spectacular increase in productivity. However, cow fertility has concomitantly severely declined. It is generally assumed that this reduction is primarily due to the negative energy balance of high-producing cows at the peak of lactation. We herein describe the fine-mapping of a major fertility QTL in Nordic Red cattle, and identify a 660-kb deletion encompassing four genes as the causative variant. We show that the deletion is a recessive embryonically lethal mutation. This probably results from the loss of RNASEH2B, which is known to cause embryonic death in mice. Despite its dramatic effect on fertility, 13%, 23% and 32% of the animals carry the deletion in Danish, Swedish and Finnish Red Cattle, respectively. To explain this, we searched for favorable effects on other traits and found that the deletion has strong positive effects on milk yield. This study demonstrates that embryonic lethal mutations account for a non-negligible fraction of the decline in fertility of domestic cattle, and that associated positive effects on milk yield may account for part of the negative genetic correlation. Our study adds to the evidence that structural variants contribute to animal phenotypic variation, and that balancing selection might be more common in livestock species than previously appreciated.

Transcriptomic and proteomic profiling of two porcine tissues using high-throughput technologies.

BackgroundThe recent development within high-throughput technologies for expression profiling has allowed for parallel analysis of transcriptomes and proteomes in biological systems such as comparative analysis of transcript and protein levels of tissue regulated genes. Until now, such studies of have only included microarray or short length sequence tags for transcript profiling. Furthermore, most comparisons of transcript and protein levels have been based on absolute expression values from within the same tissue and not relative expression values based on tissue ratios.ResultsPresented here is a novel study of two porcine tissues based on integrative analysis of data from expression profiling of identical samples using cDNA microarray, 454-sequencing and iTRAQ-based proteomics. Sequence homology identified 2.541 unique transcripts that are detectable by both microarray hybridizations and 454-sequencing of 1.2 million cDNA tags. Both transcript-based technologies showed high reproducibility between sample replicates of the same tissue, but the correlation across these two technologies was modest. Thousands of genes being differentially expressed were identified with microarray. Out of the 306 differentially expressed genes, identified by 454-sequencing, 198 (65%) were also found by microarray. The relationship between the regulation of transcript and protein levels was analyzed by integrating iTRAQ-based proteomics data. Protein expression ratios were determined for 354 genes, of which 148 could be mapped to both microarray and 454-sequencing data. A comparison of the expression ratios from the three technologies revealed that differences in transcript and protein levels across heart and muscle tissues are positively correlated.ConclusionWe show that the reproducibility within cDNA microarray and 454-sequencing is high, but that the agreement across these two technologies is modest. We demonstrate that the regulation of transcript and protein levels across identical tissue samples is positively correlated when the tissue expression ratios are used for comparison. The results presented are of interest in systems biology research in terms of integration and analysis of high-throughput expression data from mammalian tissues.

A transcriptome map of perennial ryegrass (Lolium perenne L.)

BackgroundSingle nucleotide polymorphisms (SNPs) are increasingly becoming the DNA marker system of choice due to their prevalence in the genome and their ability to be used in highly multiplexed genotyping assays. Although needed in high numbers for genome-wide marker profiles and genomics-assisted breeding, a surprisingly low number of validated SNPs are currently available for perennial ryegrass.ResultsA perennial ryegrass unigene set representing 9,399 genes was used as a reference for the assembly of 802,156 high quality reads generated by 454 transcriptome sequencing and for in silico SNP discovery. Out of more than 15,433 SNPs in 1,778 unigenes fulfilling highly stringent assembly and detection parameters, a total of 768 SNP markers were selected for GoldenGate genotyping in 184 individuals of the perennial ryegrass mapping population VrnA, a population being previously evaluated for important agronomic traits. A total of 592 (77%) of the SNPs tested were successfully called with a cluster separation above 0.9. Of these, 509 (86%) genic SNP markers segregated in the VrnA mapping population, out of which 495 were assigned to map positions. The genetic linkage map presented here comprises a total of 838 DNA markers (767 gene-derived markers) and spans 750 centi Mogan (cM) with an average marker interval distance of less than 0.9 cM. Moreover, it locates 732 expressed genes involved in a broad range of molecular functions of different biological processes in the perennial ryegrass genome.ConclusionsHere, we present an efficient approach of using next generation sequencing (NGS) data for SNP discovery, and the successful design of a 768-plex Illumina GoldenGate genotyping assay in a complex genome. The ryegrass SNPs along with the corresponding transcribed sequences represent a milestone in the establishment of genetic and genomics resources available for this species and constitute a further step towards molecular breeding strategies. Moreover, the high density genetic linkage map predominantly based on gene-associated DNA markers provides an important tool for the assignment of candidate genes to quantitative trait loci (QTL), functional genomics and the integration of genetic and physical maps in perennial ryegrass, one of the most important temperate grassland species.

SNP Discovery Using Next Generation Transcriptomic Sequencing in Atlantic Herring (Clupea harengus)

The introduction of Next Generation Sequencing (NGS) has revolutionised population genetics, providing studies of non-model species with unprecedented genomic coverage, allowing evolutionary biologists to address questions previously far beyond the reach of available resources. Furthermore, the simple mutation model of Single Nucleotide Polymorphisms (SNPs) permits cost-effective high-throughput genotyping in thousands of individuals simultaneously. Genomic resources are scarce for the Atlantic herring (Clupea harengus), a small pelagic species that sustains high revenue fisheries. This paper details the development of 578 SNPs using a combined NGS and high-throughput genotyping approach. Eight individuals covering the species distribution in the eastern Atlantic were bar-coded and multiplexed into a single cDNA library and sequenced using the 454 GS FLX platform. SNP discovery was performed by de novo sequence clustering and contig assembly, followed by the mapping of reads against consensus contig sequences. Selection of candidate SNPs for genotyping was conducted using an in silico approach. SNP validation and genotyping were performed simultaneously using an Illumina 1,536 GoldenGate assay. Although the conversion rate of candidate SNPs in the genotyping assay cannot be predicted in advance, this approach has the potential to maximise cost and time efficiencies by avoiding expensive and time-consuming laboratory stages of SNP validation. Additionally, the in silico approach leads to lower ascertainment bias in the resulting SNP panel as marker selection is based only on the ability to design primers and the predicted presence of intron-exon boundaries. Consequently SNPs with a wider spectrum of minor allele frequencies (MAFs) will be genotyped in the final panel. The genomic resources presented here represent a valuable multi-purpose resource for developing informative marker panels for population discrimination, microarray development and for population genomic studies in the wild.

Global assessment of genomic variation in cattle by genome resequencing and high-throughput genotyping

BackgroundIntegration of genomic variation with phenotypic information is an effective approach for uncovering genotype-phenotype associations. This requires an accurate identification of the different types of variation in individual genomes.ResultsWe report the integration of the whole genome sequence of a single Holstein Friesian bull with data from single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) array technologies to determine a comprehensive spectrum of genomic variation. The performance of resequencing SNP detection was assessed by combining SNPs that were identified to be either in identity by descent (IBD) or in copy number variation (CNV) with results from SNP array genotyping. Coding insertions and deletions (indels) were found to be enriched for size in multiples of 3 and were located near the N- and C-termini of proteins. For larger indels, a combination of split-read and read-pair approaches proved to be complementary in finding different signatures. CNVs were identified on the basis of the depth of sequenced reads, and by using SNP and CGH arrays.ConclusionsOur results provide high resolution mapping of diverse classes of genomic variation in an individual bovine genome and demonstrate that structural variation surpasses sequence variation as the main component of genomic variability. Better accuracy of SNP detection was achieved with little loss of sensitivity when algorithms that implemented mapping quality were used. IBD regions were found to be instrumental for calculating resequencing SNP accuracy, while SNP detection within CNVs tended to be less reliable. CNV discovery was affected dramatically by platform resolution and coverage biases. The combined data for this study showed that at a moderate level of sequencing coverage, an ensemble of platforms and tools can be applied together to maximize the accurate detection of sequence and structural variants.

A synteny-based draft genome sequence of the forage grass Lolium perenne.

Here we report the draft genome sequence of perennial ryegrass (Lolium perenne), an economically important forage and turf grass species that is widely cultivated in temperate regions worldwide. It is classified along with wheat, barley, oats and Brachypodium distachyon in the Pooideae sub-family of the grass family (Poaceae). Transcriptome data was used to identify 28,455 gene models, and we utilized macro-co-linearity between perennial ryegrass and barley, and synteny within the grass family, to establish a synteny-based linear gene order. The gametophytic self-incompatibility mechanism enables the pistil of a plant to reject self-pollen and therefore promote out-crossing. We have used the sequence assembly to characterize transcriptional changes in the stigma during pollination with both compatible and incompatible pollen. Characterization of the pollen transcriptome identified homologs to pollen allergens from a range of species, many of which were expressed to very high levels in mature pollen grains, and are potentially involved in the self-incompatibility mechanism. The genome sequence provides a valuable resource for future breeding efforts based on genomic prediction, and will accelerate the development of new varieties for more productive grasslands.

Novel Tools for Conservation Genomics: Comparing Two High-Throughput Approaches for SNP Discovery in the Transcriptome of the European Hake

The growing accessibility to genomic resources using next-generation sequencing (NGS) technologies has revolutionized the application of molecular genetic tools to ecology and evolutionary studies in non-model organisms. Here we present the case study of the European hake (Merluccius merluccius), one of the most important demersal resources of European fisheries. Two sequencing platforms, the Roche 454 FLX (454) and the Illumina Genome Analyzer (GAII), were used for Single Nucleotide Polymorphisms (SNPs) discovery in the hake muscle transcriptome. De novo transcriptome assembly into unique contigs, annotation, and in silico SNP detection were carried out in parallel for 454 and GAII sequence data. High-throughput genotyping using the Illumina GoldenGate assay was performed for validating 1,536 putative SNPs. Validation results were analysed to compare the performances of 454 and GAII methods and to evaluate the role of several variables (e.g. sequencing depth, intron-exon structure, sequence quality and annotation). Despite well-known differences in sequence length and throughput, the two approaches showed similar assay conversion rates (approximately 43%) and percentages of polymorphic loci (67.5% and 63.3% for GAII and 454, respectively). Both NGS platforms therefore demonstrated to be suitable for large scale identification of SNPs in transcribed regions of non-model species, although the lack of a reference genome profoundly affects the genotyping success rate. The overall efficiency, however, can be improved using strict quality and filtering criteria for SNP selection (sequence quality, intron-exon structure, target region score).

Genome wide allele frequency fingerprints (GWAFFs) of populations via genotyping by sequencing.

Genotyping-by-Sequencing (GBS) is an excellent tool for characterising genetic variation between plant genomes. To date, its use has been reported only for genotyping of single individuals. However, there are many applications where resolving allele frequencies within populations on a genome-wide scale would be very powerful, examples include the breeding of outbreeding species, varietal protection in outbreeding species, monitoring changes in population allele frequencies. This motivated us to test the potential to use GBS to evaluate allele frequencies within populations. Perennial ryegrass is an outbreeding species, and breeding programs are based upon selection on populations. We tested two restriction enzymes for their efficiency in complexity reduction of the perennial ryegrass genome. The resulting profiles have been termed Genome Wide Allele Frequency Fingerprints (GWAFFs), and we have shown how these fingerprints can be used to distinguish between plant populations. Even at current costs and throughput, using sequencing to directly evaluate populations on a genome-wide scale is viable. GWAFFs should find many applications, from varietal development in outbreeding species right through to playing a role in protecting plant breeders’ rights.