BACKGROUND:

Hyperpigmentation of the visceral peritoneum (HVP) has recently garnered much attention in the poultry industry because of the possible risk to the health of affected animals and the damage it causes to the appearance of commercial chicken carcasses. However, the heritable characters of HVP remain unclear. The objective of this study was to investigate the genetic parameters of HVP by genome-wide association study (GWAS) in chickens.

RESULTS:

HVP was found to be influenced by genetic factors, with a heritability score of 0.33. HVP had positive genetic correlations with growth and carcass traits, such as leg muscle weight (rg = 0.34), but had negative genetic correlations with immune traits, such as the antibody response to Newcastle disease virus (rg = -0.42). The GWAS for HVP using 39,833 single nucleotide polymorphisms indicated the genetic factors associated with HVP displayed an additive effect rather than a dominance effect. In addition, we determined that three genomic regions, involving the 50.5--54.0 Mb region of chicken (Gallus gallus) chromosome 1 (GGA1), the 58.5--60.5 Mb region of GGA1, and the 10.5--12.0 Mb region of GGA20, were strongly associated (P < 6.28 x 10-7) with HVP in chickens. Variants in these regions explained >50% of additive genetic variance for HVP. This study also confirmed that expression of BMP7, which codes for a bone morphogenetic protein and is located in one of the candidate regions, was significantly higher in the visceral peritoneum of Huiyang Beard chickens with HVP than in that of chickens without pigmentation (P < 0.05).

CONCLUSIONS:

HVP is a quantitative trait with moderate heritability. Genomic variants resulting in HVP were identified on GGA1 and GGA20, and expression of the BMP7 gene appears to be upregulated in HVP-affected chickens. Findings from this study should be used as a basis for further functional validation of candidate genes involved in HVP.

BACKGROUND:

Clostridium botulinum is a group of four physiologically and phylogenetically distinct bacteria that produce botulinum neurotoxin. While studies have characterised variability between strains of Group I (proteolytic) C. botulinum, the genetic and physiological variability and relationships between strains within Group II (non-proteolytic) C. botulinum are not well understood. In this study the genome of Group II strain C. botulinum Eklund 17B (NRP) was sequenced and used to construct a whole genome DNA microarray. This was used in a comparative genomic indexing study to compare the relatedness of 43 strains of Group II C. botulinum (14 type B, 24 type E and 5 type F). These results were compared with characteristics determined from physiological tests.

RESULTS:

Whole genome indexing showed that strains of Group II C. botulinum isolated from a wide variety of environments over more than 75 years clustered together indicating the genetic background of Group II C. botulinum is stable. Further analysis showed that strains forming type B or type F toxin are closely related with only toxin cluster genes targets being unique to either type. Strains producing type E toxin formed a separate subset. Carbohydrate fermentation tests supported the observation that type B and F strains form a separate subset to type E strains. All the type F strains and most of type B strains produced acid from amylopectin, amylose and glycogen whereas type E strains did not. However, these two subsets did not differ strongly in minimum growth temperature or maximum NaCl concentration for growth. No relationship was found between tellurite resistance and toxin type despite all the tested type B and type F strains carrying tehB, while the sequence was absent or diverged in all type E strains.

CONCLUSIONS:

Although Group II C. botulinum form a tight genetic group, genomic and physiological analysis indicates there are two distinct subsets within this group. All type B strains and type F strains are in one subset and all type E strains in the other.

BACKGROUND:

The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems.

RESULTS:

The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome.

CONCLUSIONS:

This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig's adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.

BACKGROUND:

Zebrafish embryos are transcriptionally silent until activation of the zygotic genome during the 10th cell cycle. Onset of transcription is followed by cellular and morphological changes involving cell speciation and gastrulation. Previous genome-wide surveys of transcriptional changes only assessed gene expression levels; however, recent studies have shown the necessity to map isoform-specific transcriptional changes. Here, we perform isoform discovery and quantification on transcriptome sequences from before and after zebrafish zygotic genome activation (ZGA).

RESULTS:

We identify novel isoforms and isoform switches during ZGA for genes related to cell adhesion, pluripotency and DNA methylation. Isoform switching events include alternative splicing and changes in transcriptional start sites and in 3' untranslated regions. New isoforms are identified even for well-characterized genes such as pou5f1, sall4 and dnmt1. Genes involved in cell-cell interactions such as f11r and magi1 display isoform switches with alterations of coding sequences. We also detect over 1000 transcripts that acquire a longer 3' terminal exon when transcribed by the zygote compared to their maternal transcript counterparts. ChIP-sequencing data mapped onto skipped exon events reveal a correlation between histone H3K36 trimethylation peaks and skipped exons, suggesting epigenetic marks being part of alternative splicing regulation.

CONCLUSIONS:

The novel isoforms and isoform switches reported here include regulators of transcriptional, cellular and morphological changes taking place around ZGA. Our data display an array of isoform-related functional changes and represent a valuable resource complementary to existing early embryo transcriptomes.

BACKGROUND:

Animal migration requires adaptations in morphological, physiological and behavioural traits. Several of these traits have been shown to possess a strong heritable component in birds, but little is known about their genetic architecture. Here we used 454 sequencing of brain-derived transcriptomes from two differentially migrating subspecies of the willow warbler Phylloscopus trochilus to detect genes potentially underlying traits associated with migration.

RESULTS:

The transcriptome sequencing resulted in 1.8 million reads following filtering steps. Most of the reads (84%) were successfully mapped to the genome of the zebra finch Taeniopygia gutatta. The mapped reads were situated within at least 12,101 predicted zebra finch genes, with the greatest sequencing depth in exons. Reads that were mapped to intergenic regions were generally located close to predicted genes and possibly located in uncharacterized untranslated regions (UTRs). Out of 85,000 single nucleotide polymorphisms (SNPs) with a minimum sequencing depth of eight reads from each of two subspecies-specific pools, only 55 showed high differentiation, confirming previous studies showing that most of the genetic variation is shared between the subspecies. Validation of a subset of the most highly differentiated SNPs using Sanger sequencing demonstrated that several of them also were differentiated between an independent set of individuals of each subspecies. These SNPs were clustered in two chromosome regions that are likely to be influenced by divergent selection between the subspecies and that could potentially be associated with adaptations to their different migratory strategies.

CONCLUSIONS:

Our study represents the first large-scale sequencing analysis aiming at detecting genes underlying migratory phenotypes in birds and provides new candidates for genes potentially involved in migration.

BACKGROUND:

Primula species are important early spring garden plants with a centre of diversity and speciation in the East Himalaya-Hengduan Mountains in Western China. Studies on population genetics, speciation and phylogeny of Primula have been impeded by a lack of genomic resources. In the present study, we sequenced the transcriptomes of two closely related primrose species, Primula poissonii and Primula wilsonii, using short reads on the Illumina Genome Analyzer platform.

RESULTS:

We obtained 55,284 and 55,011 contigs with N50 values of 938 and 1,085 for P. poissonii and P. wilsonii, respectively, and 6,654 pairs of putative orthologs were identified between the two species. Estimations of non-synonymous/synonymous substitution rate ratios for these orthologs indicated that 877 of the pairs may be under positive selection (Ka/Ks > 0.5), and functional enrichment analysis revealed that significant proportions of the orthologs were in the categories DNA repair, stress resistance, which may provide some hints as to how the two closely related Primula species adapted differentially to extreme environments, such as habitats characterized by aridity, high altitude and high levels of ionizing radiation. It was possible for the first time to estimate the divergence time between the radiated species pair, P. poissonii and P. wilsonii; this was found to be approximately 0.90 +/- 0.57 Mya, which falls between the Donau and Gunz glaciation in the Middle Pleistocene. Primers based on 54 pairs of orthologous SSR-containing sequences between the two Primula species were designed and verified. About half of these pairs successfully amplified for both species. Of the 959 single copy nuclear genes shared by four model plants (known as APVO genes), 111 single copy nuclear genes were verified as being present in both Primula species and exon-anchored and intron-spanned primers were designed for use.

CONCLUSION:

We characterized the transcriptomes for the two Primula species, and produced an unprecedented amount of genomic resources for these important garden plants. Evolutionary analysis of these two Primula species not only revealed a more precise divergence time, but also provided some novel insights into how differential adaptations occurred in extreme habitats. Furthermore, we developed two sets of genetic markers, single copy nuclear genes and nuclear microsatellites (EST-SSR). Both these sets of markers will facilitate studies on the genetic improvement, population genetics and phylogenetics of this rapidly adapting taxon.

BACKGROUND:

RNA-seq has shown huge potential for phylogenomic inferences in non-model organisms. However, error, incompleteness, and redundant assembled transcripts for each gene in de novo assembly of short reads cause noise in analyses and a large amount of missing data in the aligned matrix. To address these problems, we compare de novo assemblies of paired end 90 bp RNA-seq reads using Oases, Trinity, Trans-ABySS and SOAPdenovo-Trans to transcripts from genome annotation of the model plant Ricinus communis. By doing so we evaluate strategies for optimizing total gene coverage and minimizing assembly chimeras and redundancy.

RESULTS:

We found that the frequency and structure of chimeras vary dramatically among different software packages. The differences were largely due to the number of trans-self chimeras that contain repeats in the opposite direction. More than half of the total chimeras in Oases and Trinity were trans-self chimeras. Within each package, we found a trade-off between maximizing reference coverage and minimizing redundancy and chimera rate. In order to reduce redundancy, we investigated three methods: 1) using cap3 and CD-HIT-EST to combine highly similar transcripts, 2) only retaining the transcript with the highest read coverage, or removing the transcript with the lowest read coverage for each subcomponent in Trinity, and 3) filtering Oases single k-mer assemblies by number of transcripts per locus and relative transcript length, and then finding the transcript with the highest read coverage. We then utilized results from blastx against model protein sequences to effectively remove trans chimeras. After optimization, seven assembly strategies among all four packages successfully assembled 42.9--47.1% of reference genes to more than 200 bp, with a chimera rate of 0.92--2.21%, and on average 1.8--3.1 transcripts per reference gene assembled.

CONCLUSIONS:

With rapidly improving sequencing and assembly tools, our study provides a framework to benchmark and optimize performance before choosing tools or parameter combinations for analyzing short-read RNA-seq data. Our study demonstrates that choice of assembly package, k-mer sizes, post-assembly redundancy-reduction and chimera cleanup, and strand-specific RNA-seq library preparation and assembly dramatically improves gene coverage by non-redundant and non-chimeric transcripts that are optimized for downstream phylogenomic analyses.

Here we present an overview of our existing knowledge on the function of RIN4 as a regulator of plant defense and as a guardee of multiple plant R-proteins. Domain analysis of RIN4 reveals two NOI domains. The NOI domain was originally identified in a screen for nitrate induced genes. The domain is comprised of approximately 30 amino acids and contains 2 conserved motifs (PXFGXW and Y/FTXXF). The NOI gene family contains members exclusively from the plant lineage as far back as moss. In addition to the conserved NOI domain, members within the family also contain conserved C-terminal cysteine residue(s) which are sites for acylation and membrane tethering. Other than these two characteristic features, the sequence of the family of NOI-containing proteins is diverse and, with the exception of RIN4, their functions are not known. Recently published interactome data showing interactions between RIN4 and components of the exocyst complex prompt us to raise the hypothesis that RIN4 might be involved in defense associated vesicle trafficking.

BACKGROUND:

Probiotic bifidobacteria in combination with prebiotic carbohydrates have documented positive effects on human health regarding gastrointestinal disorders and improved immunity, however the selective routes of uptake remain unknown for most candidate prebiotics. The differential transcriptomes of Bifidobacterium animalis subsp. lactis Bl-04, induced by 11 potential prebiotic oligosaccharides were analyzed to identify the genetic loci involved in the uptake and catabolism of alpha- and beta-linked hexoses, and beta-xylosides.

RESULTS:

The overall transcriptome was modulated dependent on the type of glycoside (galactosides, glucosides or xylosides) utilized. Carbohydrate transporters of the major facilitator superfamily (induced by gentiobiose and beta-galacto-oligosaccharides (GOS)) and ATP-binding cassette (ABC) transporters (upregulated by cellobiose, GOS, isomaltose, maltotriose, melibiose, panose, raffinose, stachyose, xylobiose and beta-xylo-oligosaccharides) were differentially upregulated, together with glycoside hydrolases from families 1, 2, 13, 36, 42, 43 and 77. Sequence analysis of the identified solute-binding proteins that determine the specificity of ABC transporters revealed similarities in the breadth and selectivity of prebiotic utilization by bifidobacteria.

CONCLUSION:

This study identified the differential gene expression for utilization of potential prebiotics highlighting the extensive capabilities of Bifidobacterium lactis Bl-04 to utilize oligosaccharides. Results provide insights into the ability of this probiotic microbe to utilize indigestible carbohydrates in the human gastrointestinal tract.

BACKGROUND:

When faced with climate change, species must either shift their home range or adapt in situ in order to maintain optimal physiological balance with their environment. The American pika (Ochotona princeps) is a small alpine mammal with limited dispersal capacity and low tolerance for thermal stress. As a result, pikas have become an important system for examining biotic responses to changing climatic conditions. Previous research using amplified fragment length polymorphisms (AFLPs) has revealed evidence for environmental-mediated selection in O. princeps populations distributed along elevation gradients, yet the anonymity of AFLP loci and lack of available genomic resources precluded the identification of associated gene regions. Here, we harnessed next-generation sequencing technology in order to characterize the American pika transcriptome and identify a large suite of single nucleotide polymorphisms (SNPs), which can be used to elucidate elevation- and site-specific patterns of sequence variation.

RESULTS:

We constructed pooled cDNA libraries of O. princeps from high (1400m) and low (300m) elevation sites along a previously established transect in British Columbia. Transcriptome sequencing using the Roche 454 GS FLX titanium platform generated 780 million base pairs of data, which were assembled into 7,325 high coverage contigs. These contigs were used to identify 24,261 novel SNP loci. Using high resolution melt analysis, we developed 17 of these SNPs into genotyping assays, which were validated with independent DNA samples from British Columbia Canada and Oregon State USA. In addition, we detected haplotypes in the NADH dehydrogenase subunit 5 of the mitochondrial genome that were fixed and different among elevations, suggesting that this may be an informative target gene for studying the role of cellular respiration in local adaptation. We also identified contigs that were unique to each elevation, including a high elevation-specific contig that was a positive match with the hemoglobin alpha chain from the plateau pika, a species restricted to high elevation steppes in Asia. Elevation-specific contigs may represent candidate regions subject to differential levels of gene expression along this elevation gradient.

CONCLUSIONS:

To our knowledge, this is the first broad-scale, transcriptome-level study conducted within the Ochotonidae, providing novel genomic resources for studying pika ecology, behaviour and population history.