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To investigate the congenital complex vertebral malformation syndrome (CVM) in Holstein calves, two breeding studies were performed including 262 and 363 cows, respectively. Cows were selected from the Danish Cattle Database based on pedigree and insemination records. Selected cows were progeny of sires with an established heterozygous CVM genotype and pregnant after insemination with semen from another sire with heterozygous CVM genotype. Following calving the breeders should state, if the calf was normal and was requested to submit dead calves for necropsy. In both studies, significantly fewer CVM affected calves than expected were obtained; a finding probably reflecting extensive intrauterine mortality in CVM affected foetuses. The findings illustrate increased intrauterine mortality as a major potential bias in observational studies of inherited disorders.

Abstract Background Single 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. Results A 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. Conclusions Here, 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.

Abstract Background Integration 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. Results We 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. Conclusions Our 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.

Abstract Background Genetic linkage maps are necessary for mapping of mendelian traits and quantitative trait loci (QTLs). To identify the actual genes, which control these traits, a map based on gene-associated single nucleotide polymorphism (SNP) markers is highly valuable. In this study, the SNPs were genotyped in a large family material comprising more than 5,000 piglets derived from 12 Duroc boars crossed with 236 Danish Landrace/Danish Large White sows. The SNPs were identified in sequence alignments of 4,600 different amplicons obtained from the 12 boars and containing coding regions of genes derived from expressed sequence tags (ESTs) and genomic shotgun sequences. Results Linkage maps of all 18 porcine autosomes were constructed based on 456 gene-associated and six porcine EST-based SNPs. The total length of the averaged-sex whole porcine autosome was estimated to 1,711.8 cM resulting in an average SNP spacing of 3.94 cM. The female and male maps were estimated to 2,336.1 and 1,441.5 cM, respectively. The gene order was validated through comparisons to the cytogenetic and/or physical location of 203 genes, linkage to evenly spaced microsatellite markers as well as previously reported conserved synteny. A total of 330 previously unmapped genes and ESTs were mapped to the porcine autosome while ten genes were mapped to unexpected locations. Conclusion The linkage map presented here shows high accuracy in gene order. The pedigree family network as well as the large amount of meiotic events provide good reliability and make this map suitable for QTL and association studies. In addition, the linkage to the RH-map of microsatellites makes it suitable for comparison to other QTL studies.

Abstract Background The 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. Results Presented 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. Conclusion We 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.

Abstract Background The Atlantic salmon is an important aquaculture species and a very interesting species biologically, since it spawns in fresh water and develops through several stages before becoming a smolt, the stage at which it migrates to the sea to feed. The dramatic change of habitat requires physiological, morphological and behavioural changes to prepare the salmon for its new environment. These changes are called the parr-smolt transformation or smoltification, and pre-adapt the salmon for survival and growth in the marine environment. The development of hypo-osmotic regulatory ability plays an important part in facilitating the transition from rivers to the sea. The physiological mechanisms behind the developmental changes are largely unknown. An understanding of the transformation process will be vital to the future of the aquaculture industry. A knowledge of which genes are expressed prior to the smoltification process is an important basis for further studies. Results In all, 2974 unique sequences, consisting of 779 contigs and 2195 singlets, were generated for Atlantic salmon from two cDNA libraries constructed from the gills and the intestine, accession numbers [Genbank: CK877169-CK879929, CK884015-CK886537 and CN181112-CN181464]. Nearly 50% of the sequences were assigned putative functions because they showed similarity to known genes, mostly from other species, in one or more of the databases used. The Swiss-Prot database returned significant hits for 1005 sequences. These could be assigned predicted gene products, and 967 were annotated using Gene Ontology (GO) terms for molecular function, biological process and/or cellular component, employing an annotation transfer procedure. Conclusion This paper describes the construction of two cDNA libraries from pre-smolt Atlantic salmon (Salmo salar) and the subsequent EST sequencing, clustering and assigning of putative function to 1005 genes expressed in the gills and/or intestine.

Abstract Background Comparative whole genome analysis of Mammalia can benefit from the addition of more species. The pig is an obvious choice due to its economic and medical importance as well as its evolutionary position in the artiodactyls. Results We have generated ~3.84 million shotgun sequences (0.66X coverage) from the pig genome. The data are hereby released (NCBI Trace repository with center name "SDJVP", and project name "Sino-Danish Pig Genome Project") together with an initial evolutionary analysis. The non-repetitive fraction of the sequences was aligned to the UCSC human-mouse alignment and the resulting three-species alignments were annotated using the human genome annotation. Ultra-conserved elements and miRNAs were identified. The results show that for each of these types of orthologous data, pig is much closer to human than mouse is. Purifying selection has been more efficient in pig compared to human, but not as efficient as in mouse, and pig seems to have an isochore structure most similar to the structure in human. Conclusion The addition of the pig to the set of species sequenced at low coverage adds to the understanding of selective pressures that have acted on the human genome by bisecting the evolutionary branch between human and mouse with the mouse branch being approximately 3 times as long as the human branch. Additionally, the joint alignment of the shot-gun sequences to the human-mouse alignment offers the investigator a rapid way to defining specific regions for analysis and resequencing.

Mastitis, inflammation of the mammary gland, is the costliest disease in dairy cattle and a major animal welfare concern. Mastitis is usually caused by bacteria, of which staphylococci, streptococci and Escherichia coli are most frequently isolated from bovine mastitis. Bacteria activate the mammary immune system in variable ways, thereby influencing the severity of the disease. Escherichia coli is a common cause of mastitis in cattle causing both subclinical and clinical mastitis. Understanding of the molecular mechanisms that activate and regulate the host response would be central to effective prevention of mastitis and breeding of cows more resistant to mastitis. We used primary bovine mammary epithelial cell cultures extracted noninvasively from bovine milk samples to monitor the cellular responses to Escherichia coli challenge. Differences in gene expression between control and challenged cells were studied by total RNA-sequencing at two time points post-challenge. In total, 150 and 440 (P adj  < 0.05) differentially expressed genes were identified at 3 h and 24 h post-challenge, respectively. The differentially expressed genes were mostly upregulated at 3 h (141/150) and 24 h (424/440) post-challenge. Our results are in line with known effects of E. coli infection, with a strong early inflammatory response mediated by pathogen receptor families. Among the most significantly enriched early KEGG pathways were the TNF signalling pathway, the cytokine-cytokine receptor interaction, and the NF-kappa B signalling pathway. At 24 h post-challenge, most significantly enriched were the Influenza A, the NOD-like receptor signalling, and the IL-17 signaling pathway., (© 2024. The Author(s).)

Protein sequences of members of the plasminogen activation system are present throughout the entire vertebrate phylum. This important and well-described proteolytic cascade is governed by numerous protease-substrate and protease-inhibitor interactions whose conservation is crucial to maintaining unchanged protein function throughout evolution. The pressure to preserve protein-protein interactions may lead to either co-conservation or covariation of binding interfaces. Here, we combined covariation analysis and structure-based prediction to analyze the binding interfaces of urokinase (uPA):plasminogen activator inhibitor-1 (PAI-1) and uPA:plasminogen complexes. We detected correlated variation between the S3-pocket-lining residues of uPA and the P3 residue of both PAI-1 and plasminogen. These residues are known to form numerous polar interactions in the human uPA:PAI-1 Michaelis complex. To test the effect of mutations that correlate with each other and have occurred during mammalian diversification on protein-protein interactions, we produced uPA, PAI-1, and plasminogen from human and zebrafish to represent mammalian and nonmammalian orthologs. Using single amino acid point substitutions in these proteins, we found that the binding interfaces of uPA:plasminogen and uPA:PAI-1 may have coevolved to maintain tight interactions. Moreover, we conclude that although the interaction areas between protease-substrate and protease-inhibitor are shared, the two interactions are mechanistically different. Compared with a protease cleaving its natural substrate, the interaction between a protease and its inhibitor is more complex and involves a more fine-tuned mechanism. Understanding the effects of evolution on specific protein interactions may help further pharmacological interventions of the plasminogen activation system and other proteolytic systems., (© 2019 Jendroszek et al.)

Background: The plasminogen (PLG) activation system is composed by a series of serine proteases, inhibitors and several binding proteins, which together control the temporal and spatial generation of the active serine protease plasmin. As this proteolytic system plays a central role in human physiology and pathophysiology it has been extensively studied in mammals. The serine proteases of this system are believed to originate from an ancestral gene by gene duplications followed by domain gains and deletions. However, the identification of ancestral forms in primitive chordates supporting these theories remains elusive. In addition, evolutionary studies of the non-proteolytic members of this system are scarce., Results: Our phylogenetic analyses place lamprey PLG at the root of the vertebrate PLG-group, while lamprey PLG-related growth factors represent the ancestral forms of the jawed-vertebrate orthologues. Furthermore, we find that the earliest putative orthologue of the PLG activator group is the hyaluronan binding protein 2 (HABP2) gene found in lampreys. The prime plasminogen activators (tissue- and urokinase-type plasminogen activator, tPA and uPA) first occur in cartilaginous fish and phylogenetic analyses confirm that all orthologues identified compose monophyletic groups to their mammalian counterparts. Cartilaginous fishes exhibit the most ancient vitronectin of all vertebrates, while plasminogen activator inhibitor 1 (PAI-1) appears for the first time in cartilaginous fishes and is conserved in the rest of jawed vertebrate clades. PAI-2 appears for the first time in the common ancestor of reptiles and mammals, and represents the latest appearing plasminogen activator inhibitor. Finally, we noted that the urokinase-type plasminogen activator receptor (uPAR)-and three-LU domain containing genes in general-occurred later in evolution and was first detectable after coelacanths., Conclusions: This study identifies several primitive orthologues of the mammalian plasminogen activation system. These ancestral forms provide clues to the origin and diversification of this enzyme system. Further, the discovery of several members-hitherto unknown in mammals-provide new perspectives on the evolution of this important enzyme system.

MicroRNAs (miRNA) are key modulators of gene expression and so act as putative fine-tuners of complex phenotypes. Here, we hypothesized that causal variants of complex traits are enriched in miRNAs and miRNA-target networks. First, we conducted a genome-wide association study (GWAS) for seven functional and milk production traits using imputed sequence variants (13~15 million) and >10,000 animals from three dairy cattle breeds, i.e., Holstein (HOL), Nordic red cattle (RDC) and Jersey (JER). Second, we analyzed for enrichments of association signals in miRNAs and their miRNA-target networks. Our results demonstrated that genomic regions harboring miRNA genes were significantly (P < 0.05) enriched with GWAS signals for milk production traits and mastitis, and that enrichments within miRNA-target gene networks were significantly higher than in random gene-sets for the majority of traits. Furthermore, most between-trait and across-breed correlations of enrichments with miRNA-target networks were significantly greater than with random gene-sets, suggesting pleiotropic effects of miRNAs. Intriguingly, genes that were differentially expressed in response to mammary gland infections were significantly enriched in the miRNA-target networks associated with mastitis. All these findings were consistent across three breeds. Collectively, our observations demonstrate the importance of miRNAs and their targets for the expression of complex traits.

The American mink (Neovison vison) is a semiaquatic species of mustelid native to North America. It's an important animal for the fur industry. Many efforts have been made to locate genes influencing fur quality and color, but this search has been impeded by the lack of a reference genome. Here we present the first draft genome of mink. In our study, two mink individuals were sequenced by Illumina sequencing with 797 Gb sequence generated. Assembly yielded 7,175 scaffolds with an N50 of 6.3 Mb and length of 2.4 Gb including gaps. Repeat sequences constitute around 31% of the genome, which is lower than for dog and cat genomes. The alignments of mink, ferret and dog genomes help to illustrate the chromosomes rearrangement. Gene annotation identified 21,053 protein-coding sequences present in mink genome. The reference genome's structure is consistent with the microsatellite-based genetic map. Mapping of well-studied genes known to be involved in coat quality and coat color, and previously located fur quality QTL provide new knowledge about putative candidate genes for fur traits. The draft genome shows great potential to facilitate genomic research towards improved breeding for high fur quality animals and strengthen our understanding on evolution of Carnivora.

Plasminogen activator inhibitor type 1 (PAI-1) is a central regulator of fibrinolysis and tissue remodelling. PAI-1 belongs to the serpin superfamily and unlike other inhibitory serpins undergoes a spontaneous inactivation process under physiological conditions, termed latency transition. During latency transition the solvent exposed reactive centre loop is inserted into the central β-sheet A of the molecule, and is no longer accessible to reaction with the protease. More than three decades of research on mammalian PAI-1 has not been able to clarify the evolutionary advantage and physiological relevance of latency transition. In order to study the origin of PAI-1 latency transition, we produced PAI-1 from Spiny dogfish shark (Squalus acanthias) and African lungfish (Protopterus sp.), which represent central species in the evolution of vertebrates. Although human PAI-1 and the non-mammalian PAI-1 variants share only approximately 50 % sequence identity, our results showed that all tested PAI-1 variants undergo latency transition with a similar rate. Since the functional stability of PAI-1 can be greatly increased by substitution of few amino acid residues, we conclude that the ability to undergo latency transition must have been a specific selection criterion for the evolution of PAI-1. It appears that all PAI-1 molecules must harbour latency transition to fulfil their physiological function, stressing the importance to further pursue a complete understanding of this molecular phenomenon with possible implication to pharmacological intervention. Our results provide the next step in understanding how the complete role of this important protease inhibitor evolved along with the fibrinolytic system.

The spiny dogfish shark (Squalus acanthias) is one of the most commonly used cartilaginous fishes in biological research, especially in the fields of nitrogen metabolism, ion transporters and osmoregulation. Nonetheless, transcriptomic data for this organism is scarce. In the present study, a multi-tissue RNA-seq experiment and de novo transcriptome assembly was performed in four different spiny dogfish tissues (brain, liver, kidney and ovary), providing an annotated sequence resource. The characterization of the transcriptome greatly increases the scarce sequence information for shark species. Reads were assembled with the Trinity de novo assembler both within each tissue and across all tissues combined resulting in 362,690 transcripts in the combined assembly which represent 289,515 Trinity genes. BUSCO analysis determined a level of 87% completeness for the combined transcriptome. In total, 123,110 proteins were predicted of which 78,679 and 83,164 had significant hits against the SwissProt and Uniref90 protein databases, respectively. Additionally, 61,215 proteins aligned to known protein domains, 7,208 carried a signal peptide and 15,971 possessed at least one transmembrane region. Based on the annotation, 81,582 transcripts were assigned to gene ontology terms and 42,078 belong to known clusters of orthologous groups (eggNOG). To demonstrate the value of our molecular resource, we show that the improved transcriptome data enhances the current possibilities of osmoregulation research in spiny dogfish by utilizing the novel gene and protein annotations to investigate a set of genes involved in urea synthesis and urea, ammonia and water transport, all of them crucial in osmoregulation. We describe the presence of different gene copies and isoforms of key enzymes involved in this process, including arginases and transporters of urea and ammonia, for which sequence information is currently absent in the databases for this model species. The transcriptome assemblies and the derived annotations generated in this study will support the ongoing research for this particular animal model and provides a new molecular tool to assist biological research in cartilaginous fishes.

Background: Over the last few years, continuous development of high-throughput sequencing platforms and sequence analysis tools has facilitated reliable identification and characterization of genetic variants in many cattle breeds. Deep sequencing of entire genomes within a cattle breed that has not been thoroughly investigated would be imagined to discover functional variants that are underlying phenotypic differences. Here, we sequenced to a high coverage the Danish Holstein cattle breed to detect and characterize single nucleotide polymorphisms (SNPs), insertion/deletions (Indels), and loss-of-function (LoF) variants in protein-coding genes in order to provide a comprehensive resource for subsequent detection of causal variants for recessive traits., Results: We sequenced four genetically unrelated Danish Holstein cows with a mean coverage of 27X using an Illumina Hiseq 2000. Multi-sample SNP calling identified 10,796,794 SNPs and 1,295,036 indels whereof 482,835 (4.5 %) SNPs and 231,359 (17.9 %) indels were novel. A comparison between sequencing-derived SNPs and genotyping from the BovineHD BeadChip revealed a concordance rate of 99.6-99.8 % for homozygous SNPs and 93.3-96.5 % for heterozygous SNPs. Annotation of the SNPs discovered 74,886 SNPs and 1937 indels affecting coding sequences with 2145 being LoF mutations. The frequency of LoF variants differed greatly across the genome, a hot spot with a strikingly high density was observed in a 6 Mb region on BTA18. LoF affected genes were enriched for functional categories related to olfactory reception and underrepresented for genes related to key cellular constituents and cellular and biological process regulation. Filtering using sequence derived genotype data for 288 Holstein animals from the 1000 bull genomes project removing variants containing homozygous individuals retained 345 of the LoF variants as putatively deleterious. A substantial number of the putative deleterious LoF variants had a minor allele frequency >0.05 in the 1000 bull genomes data set., Conclusions: Deep sequencing of Danish Holstein genomes enabled us to identify 12.1 million variants. An investigation into LoF variants discovered a set of variants predicted to disrupt protein-coding genes. This catalog of variants will be a resource for future studies to understand variation underlying important phenotypes, particularly recessively inherited lethal phenotypes.

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., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

We announce the draft genome sequence of Clostridium tyrobutyricum strain DIVETGP. This strain was isolated from cow's milk used for Grana Padano cheese production. The genome was obtained using Illumina HiSeq technology and comprises 45 contigs for 3,018,999 bp, with a G+C content of 30.8%., (Copyright © 2015 Soggiu et al.)

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.

Background: The assembly of the bread wheat genome sequence is challenging due to allohexaploidy and extreme repeat content (>80%). Isolation of single chromosome arms by flow sorting can be used to overcome the polyploidy problem, but the repeat content cause extreme assembly fragmentation even at a single chromosome level. Long jump paired sequencing data (mate pairs) can help reduce assembly fragmentation by joining multiple contigs into single scaffolds. The aim of this work was to assess how mate pair data generated from multiple displacement amplified DNA of flow-sorted chromosomes affect assembly fragmentation of shotgun assemblies of the wheat chromosomes., Results: Three mate pair (MP) libraries (2 Kb, 3 Kb, and 5 Kb) were sequenced to a total coverage of 89x and 64x for the short and long arm of chromosome 7B, respectively. Scaffolding using SSPACE improved the 7B assembly contiguity and decreased gene space fragmentation, but the degree of improvement was greatly affected by scaffolding stringency applied. At the lowest stringency the assembly N50 increased by ~7 fold, while at the highest stringency N50 was only increased by ~1.5 fold. Furthermore, a strong positive correlation between estimated scaffold reliability and scaffold assembly stringency was observed. A 7BS scaffold assembly with reduced MP coverage proved that assembly contiguity was affected only to a small degree down to ~50% of the original coverage., Conclusion: The effect of MP data integration into pair end shotgun assemblies of wheat chromosome was moderate; possibly due to poor contig assembly contiguity, the extreme repeat content of wheat, and the use of amplified chromosomal DNA for MP library construction.

Background: Perennial ryegrass (Lolium perenne L.) is one of the most important forage and turf grass species of temperate regions worldwide. Its mitochondrial genome is inherited maternally and contains genes that can influence traits of agricultural importance. Moreover, the DNA sequence of mitochondrial genomes has been established and compared for a large number of species in order to characterize evolutionary relationships. Therefore, it is crucial to understand the organization of the mitochondrial genome and how it varies between and within species. Here, we report the first de novo assembly and annotation of the complete mitochondrial genome from perennial ryegrass., Results: Intact mitochondria from perennial ryegrass leaves were isolated and used for mtDNA extraction. The mitochondrial genome was sequenced to a 167-fold coverage using the Roche 454 GS-FLX Titanium platform, and assembled into a circular master molecule of 678,580 bp. A total of 34 proteins, 14 tRNAs and 3 rRNAs are encoded by the mitochondrial genome, giving a total gene space of 48,723 bp (7.2%). Moreover, we identified 149 open reading frames larger than 300 bp and covering 67,410 bp (9.93%), 250 SSRs, 29 tandem repeats, 5 pairs of large repeats, and 96 pairs of short inverted repeats. The genes encoding subunits of the respiratory complexes - nad1 to nad9, cob, cox1 to cox3 and atp1 to atp9 - all showed high expression levels both in absolute numbers and after normalization., Conclusions: The circular master molecule of the mitochondrial genome from perennial ryegrass presented here constitutes an important tool for future attempts to compare mitochondrial genomes within and between grass species. Our results also demonstrate that mitochondria of perennial ryegrass contain genes crucial for energy production that are well conserved in the mitochondrial genome of monocotyledonous species. The expression analysis gave us first insights into the transcriptome of these mitochondrial genes in perennial ryegrass.