Twelve cDNA libraries from two species of catfish have been sequenced, resulting in the generation of nearly 500,000 ESTs.
Through the Community Sequencing Program, a catfish EST sequencing project was carried out through a collaboration between the catfish research community and the Department of Energy's Joint Genome Institute. Prior to this project, only a limited EST resource from catfish was available for the purpose of SNP identification.
A total of 438,321 quality ESTs were generated from 8 channel catfish (Ictalurus punctatus) and 4 blue catfish (Ictalurus furcatus) libraries, bringing the number of catfish ESTs to nearly 500,000. Assembly of all catfish ESTs resulted in 45,306 contigs and 66,272 singletons. Over 35% of the unique sequences had significant similarities to known genes, allowing the identification of 14,776 unique genes in catfish. Over 300,000 putative SNPs have been identified, of which approximately 48,000 are high-quality SNPs identified from contigs with at least four sequences and the minor allele presence of at least two sequences in the contig. The EST resource should be valuable for identification of microsatellites, genome annotation, large-scale expression analysis, and comparative genome analysis.
This project generated a large EST resource for catfish that captured the majority of the catfish transcriptome. The parallel analysis of ESTs from two closely related Ictalurid catfishes should also provide powerful means for the evaluation of ancient and recent gene duplications, and for the development of high-density microarrays in catfish. The inter- and intra-specific SNPs identified from all catfish EST dataset assembly will greatly benefit the catfish introgression breeding program and whole genome association studies.
The fish gill, as one of the mucosal barriers, plays an important role in mucosal immune response. The fish swimbladder functions for regulating buoyancy. The fish swimbladder has long been postulated as a homologous organ of the tetrapod lung, but the molecular evidence is scarce. In order to provide new information that is complementary to gill immune genes, initiate new research directions concerning the genetic basis of the gill immune response and understand the molecular function of swimbladder as well as its relationship with lungs, transcriptome analysis of the fugu Takifugu rubripes gill and swimbladder was carried out by RNA-Seq. Approximately 55,061,524 and 44,736,850 raw sequence reads from gill and swimbladder were generated, respectively. Gene ontology (GO) and KEGG pathway analysis revealed diverse biological functions and processes. Transcriptome comparison between gill and swimbladder resulted in 3,790 differentially expressed genes, of which 1,520 were up-regulated in the swimbladder while 2,270 were down-regulated. In addition, 406 up regulated isoforms and 296 down regulated isoforms were observed in swimbladder in comparison to gill. By the gene enrichment analysis, the three immune-related pathways and 32 immune-related genes in gill were identified. In swimbladder, five pathways including 43 swimbladder-enriched genes were identified. This work should set the foundation for studying immune-related genes for the mucosal immunity and provide genomic resources to study the relatedness of the fish swimbladder and mammalian lung.
Comparative mapping is a powerful tool to study evolution of genomes. It allows transfer of genome information from the well-studied model species to non-model species. Catfish is an economically important aquaculture species in United States. A large amount of genome resources have been developed from catfish including genetic linkage maps, physical maps, BAC end sequences (BES), integrated linkage and physical maps using BES-derived markers, physical map contig-specific sequences, and draft genome sequences. Application of such genome resources should allow comparative analysis at the genome scale with several other model fish species.
In this study, we conducted whole genome comparative analysis between channel catfish and four model fish species with fully sequenced genomes, zebrafish, medaka, stickleback and Tetraodon. A total of 517 Mb draft genome sequences of catfish were anchored to its genetic linkage map, which accounted for 62% of the total draft genome sequences. Based on the location of homologous genes, homologous chromosomes were determined among catfish and the four model fish species. A large number of conserved syntenic blocks were identified. Analysis of the syntenic relationships between catfish and the four model fishes supported that the catfish genome is most similar to the genome of zebrafish.
The organization of the catfish genome is similar to that of the four teleost species, zebrafish, medaka, stickleback, and Tetraodon such that homologous chromosomes can be identified. Within each chromosome, extended syntenic blocks were evident, but the conserved syntenies at the chromosome level involve extensive inter-chromosomal and intra-chromosomal rearrangements. This whole genome comparative map should facilitate the whole genome assembly and annotation in catfish, and will be useful for genomic studies of various other fish species.
Catfish; Genome; Comparative mapping; Linkage mapping; Conserved synteny
The dioecious system is relatively rare in plants. Shrub willow is an annual flowering dioecious woody plant, and possesses many characteristics that lend it as a great model for tracking the missing pieces of sex determination evolution. To gain a global view of the genes differentially expressed in the male and female shrub willows and to develop a database for further studies, we performed a large-scale transcriptome sequencing of flower buds which were separately collected from two types of sexes.
Totally, 1,201,931 high quality reads were obtained, with an average length of 389 bp and a total length of 467.96 Mb. The ESTs were assembled into 29,048 contigs, and 132,709 singletons. These unigenes were further functionally annotated by comparing their sequences to different proteins and functional domain databases and assigned with Gene Ontology (GO) terms. A biochemical pathway database containing 291 predicted pathways was also created based on the annotations of the unigenes. Digital expression analysis identified 806 differentially expressed genes between the male and female flower buds. And 33 of them located on the incipient sex chromosome of Salicaceae, among which, 12 genes might involve in plant sex determination empirically. These genes were worthy of special notification in future studies.
In this study, a large number of EST sequences were generated from the flower buds of a male and a female shrub willow. We also reported the differentially expressed genes between the two sex-type flowers. This work provides valuable information and sequence resources for uncovering the sex determining genes and for future functional genomics analysis of Salicaceae spp.
Calpains, a superfamily of intracellular calcium-dependent cysteine proteases, are involved in the cytoskeletal remodeling and wasting of skeletal muscle. Calpains are generated as inactive proenzymes which are activated by N-terminal autolysis induced by calcium-ions.
In this study, we characterized the full-length cDNA sequences of three calpain genes, clpn1, clpn2, and clpn3 in channel catfish, and assessed the effect of nutrient restriction and subsequent re-feeding on the expression of these genes in skeletal muscle. The clpn1 cDNA sequence encodes a protein of 704 amino acids, Clpn2 of 696 amino acids, and Clpn3 of 741 amino acids. Phylogenetic analysis of deduced amino acid sequences indicate that catfish Clpn1 and Clpn2 share a sequence similarity of 61%; catfish Clpn1 and Clpn3 of 48%, and Clpn2 and Clpn3 of only 45%. The domain structure architectures of all three calpain genes in channel catfish are similar to those of other vertebrates, further supported by strong bootstrap values during phylogenetic analyses. Starvation of channel catfish (average weight, 15–20 g) for 35 days influenced the expression of clpn1 (2.3-fold decrease, P<0.05), clpn2 (1.3-fold increase, P<0.05), and clpn3 (13.0-fold decrease, P<0.05), whereas the subsequent refeeding did not change the expression of these genes as measured by quantitative real-time PCR analysis. Calpain catalytic activity in channel catfish skeletal muscle showed significant differences only during the starvation period, with a 1.2- and 1.4- fold increase (P<0.01) after 17 and 35 days of starvation, respectively.
We have assessed that fasting and refeeding may provide a suitable experimental model to provide us insight into the role of calpains during fish muscle atrophy and how they respond to changes in nutrient supply.
Construction of high-density genetic linkage maps is crucially important for quantitative trait loci (QTL) studies, and they are more useful when integrated with physical maps. Such integrated maps are valuable genome resources for fine mapping of QTL, comparative genomics, and accurate and efficient whole-genome assembly. Previously, we established both linkage maps and a physical map for channel catfish, Ictalurus punctatus, the dominant aquaculture species in the United States. Here we added 2030 BAC end sequence (BES)-derived microsatellites from 1481 physical map contigs, as well as markers from singleton BES, ESTs, anonymous microsatellites, and SNPs, to construct a second-generation linkage map. Average marker density across the 29 linkage groups reached 1.4 cM/marker. The increased marker density highlighted variations in recombination rates within and among catfish chromosomes. This work effectively anchored 44.8% of the catfish BAC physical map contigs, covering ∼52.8% of the genome. The genome size was estimated to be 2546 cM on the linkage map, and the calculated physical distance per centimorgan was 393 Kb. This integrated map should enable comparative studies with teleost model species as well as provide a framework for ordering and assembling whole-genome scaffolds.
catfish; linkage map; physical map; genome; map integration
Single nucleotide polymorphisms (SNPs) have become the marker of choice for genome-wide association studies. In order to provide the best genome coverage for the analysis of performance and production traits, a large number of relatively evenly distributed SNPs are needed. Gene-associated SNPs may fulfill these requirements of large numbers and genome wide distribution. In addition, gene-associated SNPs could themselves be causative SNPs for traits. The objective of this project was to identify large numbers of gene-associated SNPs using high-throughput next generation sequencing.
Transcriptome sequencing was conducted for channel catfish and blue catfish using Illumina next generation sequencing technology. Approximately 220 million reads (15.6 Gb) for channel catfish and 280 million reads (19.6 Gb) for blue catfish were obtained by sequencing gene transcripts derived from various tissues of multiple individuals from a diverse genetic background. A total of over 35 billion base pairs of expressed short read sequences were generated. Over two million putative SNPs were identified from channel catfish and almost 2.5 million putative SNPs were identified from blue catfish. Of these putative SNPs, a set of filtered SNPs were identified including 342,104 intra-specific SNPs for channel catfish, 366,269 intra-specific SNPs for blue catfish, and 420,727 inter-specific SNPs between channel catfish and blue catfish. These filtered SNPs are distributed within 16,562 unique genes in channel catfish and 17,423 unique genes in blue catfish.
For aquaculture species, transcriptome analysis of pooled RNA samples from multiple individuals using Illumina sequencing technology is both technically efficient and cost-effective for generating expressed sequences. Such an approach is most effective when coupled to existing EST resources generated using traditional sequencing approaches because the reference ESTs facilitate effective assembly of the expressed short reads. When multiple individuals with different genetic backgrounds are used, RNA-Seq is very effective for the identification of SNPs. The SNPs identified in this report will provide a much needed resource for genetic studies in catfish and will contribute to the development of a high-density SNP array. Validation and testing of these SNPs using SNP arrays will form the material basis for genome association studies and whole genome-based selection in catfish.
Genome annotation projects, gene functional studies, and phylogenetic analyses for a given organism all greatly benefit from access to a validated full-length cDNA resource. While increasingly common in model species, full-length cDNA resources in aquaculture species are scarce.
Methodology and Principal Findings
Through in silico analysis of catfish (Ictalurus spp.) ESTs, a total of 10,037 channel catfish and 7,382 blue catfish cDNA clones were identified as potentially encoding full-length cDNAs. Of this set, a total of 1,169 channel catfish and 933 blue catfish full-length cDNA clones were selected for re-sequencing to provide additional coverage and ensure sequence accuracy. A total of 1,745 unique gene transcripts were identified from the full-length cDNA set, including 1,064 gene transcripts from channel catfish and 681gene transcripts from blue catfish, with 416 transcripts shared between the two closely related species. Full-length sequence characteristics (ortholog conservation, UTR length, Kozak sequence, and conserved motifs) of the channel and blue catfish were examined in detail. Comparison of gene ontology composition between full-length cDNAs and all catfish ESTs revealed that the full-length cDNA set is representative of the gene diversity encoded in the catfish transcriptome.
This study describes the first catfish full-length cDNA set constructed from several cDNA libraries. The catfish full-length cDNA sequences, and data gleaned from sequence characteristics analysis, will be a valuable resource for ongoing catfish whole-genome sequencing and future gene-based studies of function and evolution in teleost fishes.
Recent studies have found that copy number variations (CNVs) are widespread in human and animal genomes. CNVs are a significant source of genetic variation, and have been shown to be associated with phenotypic diversity. However, the effect of CNVs on genetic variation in horses is not well understood. In the present study, CNVs in 6 different breeds of mare horses, Mongolia horse, Abaga horse, Hequ horse and Kazakh horse (all plateau breeds) and Debao pony and Thoroughbred, were determined using aCGH. In total, seven hundred CNVs were identified ranging in size from 6.1 Kb to 0.57 Mb across all autosomes, with an average size of 43.08 Kb and a median size of 15.11 Kb. By merging overlapping CNVs, we found a total of three hundred and fifty-three CNV regions (CNVRs). The length of the CNVRs ranged from 6.1 Kb to 1.45 Mb with average and median sizes of 38.49 Kb and 13.1 Kb. Collectively, 13.59 Mb of copy number variation was identified among the horses investigated and accounted for approximately 0.61% of the horse genome sequence. Five hundred and eighteen annotated genes were affected by CNVs, which corresponded to about 2.26% of all horse genes. Through the gene ontology (GO), genetic pathway analysis and comparison of CNV genes among different breeds, we found evidence that CNVs involving 7 genes may be related to the adaptation to severe environment of these plateau horses. This study is the first report of copy number variations in Chinese horses, which indicates that CNVs are ubiquitous in the horse genome and influence many biological processes of the horse. These results will be helpful not only in mapping the horse whole-genome CNVs, but also to further research for the adaption to the high altitude severe environment for plateau horses.
Houttuynia cordata Thunb. is an important traditional medical herb in China and other Asian countries, with high medicinal and economic value. However, a lack of available genomic information has become a limitation for research on this species. Thus, we carried out high-throughput transcriptomic sequencing of H. cordata to generate an enormous transcriptome sequence dataset for gene discovery and molecular marker development.
Illumina paired-end sequencing technology produced over 56 million sequencing reads from H. cordata mRNA. Subsequent de novo assembly yielded 63,954 unigenes, 39,982 (62.52%) and 26,122 (40.84%) of which had significant similarity to proteins in the NCBI nonredundant protein and Swiss-Prot databases (E-value <10−5), respectively. Of these annotated unigenes, 30,131 and 15,363 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. In addition, 24,434 (38.21%) unigenes were mapped onto 128 pathways using the KEGG pathway database and 17,964 (44.93%) unigenes showed homology to Vitis vinifera (Vitaceae) genes in BLASTx analysis. Furthermore, 4,800 cDNA SSRs were identified as potential molecular markers. Fifty primer pairs were randomly selected to detect polymorphism among 30 samples of H. cordata; 43 (86%) produced fragments of expected size, suggesting that the unigenes were suitable for specific primer design and of high quality, and the SSR marker could be widely used in marker-assisted selection and molecular breeding of H. cordata in the future.
This is the first application of Illumina paired-end sequencing technology to investigate the whole transcriptome of H. cordata and to assemble RNA-seq reads without a reference genome. These data should help researchers investigating the evolution and biological processes of this species. The SSR markers developed can be used for construction of high-resolution genetic linkage maps and for gene-based association analyses in H. cordata. This work will enable future functional genomic research and research into the distinctive active constituents of this genus.
The application of RNA-seq has accelerated gene expression profiling and identification of gene-associated SNPs in many species. However, the integrated studies of gene expression along with SNP mapping have been lacking. Coupling of RNA-seq with bulked segregant analysis (BSA) should allow correlation of expression patterns and associated SNPs with the phenotypes.
In this study, we demonstrated the use of bulked segregant RNA-seq (BSR-Seq) for the analysis of differentially expressed genes and associated SNPs with disease resistance against enteric septicemia of catfish (ESC). A total of 1,255 differentially expressed genes were found between resistant and susceptible fish. In addition, 56,419 SNPs residing on 4,304 unique genes were identified as significant SNPs between susceptible and resistant fish. Detailed analysis of these significant SNPs allowed differentiation of significant SNPs caused by genetic segregation and those caused by allele-specific expression. Mapping of the significant SNPs, along with analysis of differentially expressed genes, allowed identification of candidate genes underlining disease resistance against ESC disease.
This study demonstrated the use of BSR-Seq for the identification of genes involved in disease resistance against ESC through expression profiling and mapping of significantly associated SNPs. BSR-Seq is applicable to analysis of genes underlining various performance and production traits without significant investment in the development of large genotyping platforms such as SNP arrays.
Bulk segregant analysis; RNA-seq; Disease resistance; Catfish; Allele-specific expression
An increasing awareness of the vulnerability of sharks to exploitation by shark finning has contributed to a growing concern about an unsustainable shark fishery. Taiwan’s fleet has the 4th largest shark catch in the world, accounting for almost 6% of the global figures. Revealing the diversity of sharks consumed by Taiwanese is important in designing conservation plans. However, fins make up less than 5% of the total body weight of a shark, and their bodies are sold as filets in the market, making it difficult or impossible to identify species using morphological traits.
In the present study, we adopted a DNA barcoding technique using a 391-bp fragment of the mitochondrial cytochrome oxidase I (COI) gene to examine the diversity of shark filets and fins collected from markets and restaurants island-wide in Taiwan.
Amongst the 548 tissue samples collected and sequenced, 20 major clusters were apparent by phylogenetic analyses, each of them containing individuals belonging to the same species (most with more than 95% bootstrap values), corresponding to 20 species of sharks. Additionally, Alopias pelagicus, Carcharhinus falciformis, Isurus oxyrinchus, and Prionace glauca consisted of 80% of the samples we collected, indicating that these species might be heavily consumed in Taiwan. Approximately 5% of the tissue samples used in this study were identified as species listed in CITES Appendix II, including two species of Sphyrna, C. longimanus and Carcharodon carcharias.
DNA barcoding provides an alternative method for understanding shark species composition when species-specific data is unavailable. Considering the global population decline, stock assessments of Appendix II species and highly consumed species are needed to accomplish the ultimate goal of shark conservation.
The intensive selection programs for milk made possible by mass artificial insemination increased the similarity among the genomes of North American (NA) Holsteins tremendously since the 1960s. This migration of elite alleles has caused certain regions of the genome to have runs of homozygosity (ROH) occasionally spanning millions of continuous base pairs at a specific locus. In this study, genome signatures of artificial selection in NA Holsteins born between 1953 and 2008 were identified by comparing changes in ROH between three distinct groups under different selective pressure for milk production. The ROH regions were also used to estimate the inbreeding coefficients. The comparisons of genomic autozygosity between groups selected or unselected since 1964 for milk production revealed significant differences with respect to overall ROH frequency and distribution. These results indicate selection has increased overall autozygosity across the genome, whereas the autozygosity in an unselected line has not changed significantly across most of the chromosomes. In addition, ROH distribution was more variable across the genomes of selected animals in comparison to a more even ROH distribution for unselected animals. Further analysis of genome-wide autozygosity changes and the association between traits and haplotypes identified more than 40 genomic regions under selection on several chromosomes (Chr) including Chr 2, 7, 16 and 20. Many of these selection signatures corresponded to quantitative trait loci for milk, fat, and protein yield previously found in contemporary Holsteins.
Along with the rapid advances of the nextgen sequencing technologies, more and more species are added to the list of organisms whose whole genomes are sequenced. However, the assembled draft genome of many organisms consists of numerous small contigs, due to the short length of the reads generated by nextgen sequencing platforms. In order to improve the assembly and bring the genome contigs together, more genome resources are needed. In this study, we developed a strategy to generate a valuable genome resource, physical map contig-specific sequences, which are randomly distributed genome sequences in each physical contig. Two-dimensional tagging method was used to create specific tags for 1,824 physical contigs, in which the cost was dramatically reduced. A total of 94,111,841 100-bp reads and 315,277 assembled contigs are identified containing physical map contig-specific tags. The physical map contig-specific sequences along with the currently available BAC end sequences were then used to anchor the catfish draft genome contigs. A total of 156,457 genome contigs (~79% of whole genome sequencing assembly) were anchored and grouped into 1,824 pools, in which 16,680 unique genes were annotated. The physical map contig-specific sequences are valuable resources to link physical map, genetic linkage map and draft whole genome sequences, consequently have the capability to improve the whole genome sequences assembly and scaffolding, and improve the genome-wide comparative analysis as well. The strategy developed in this study could also be adopted in other species whose whole genome assembly is still facing a challenge.
The regulatory role of miRNA in gene expression is an emerging hot new topic in the control of hypometabolism. Sea cucumber aestivation is a complicated physiological process that includes obvious hypometabolism as evidenced by a decrease in the rates of oxygen consumption and ammonia nitrogen excretion, as well as a serious degeneration of the intestine into a very tiny filament. To determine whether miRNAs play regulatory roles in this process, the present study analyzed profiles of miRNA expression in the intestine of the sea cucumber (Apostichopus japonicus), using Solexa deep sequencing technology. We identified 308 sea cucumber miRNAs, including 18 novel miRNAs specific to sea cucumber. Animals sampled during deep aestivation (DA) after at least 15 days of continuous torpor, were compared with animals from a non-aestivation (NA) state (animals that had passed through aestivation and returned to the active state). We identified 42 differentially expressed miRNAs [RPM (reads per million) >10, |FC| (|fold change|) ≥1, FDR (false discovery rate) <0.01] during aestivation, which were validated by two other miRNA profiling methods: miRNA microarray and real-time PCR. Among the most prominent miRNA species, miR-200-3p, miR-2004, miR-2010, miR-22, miR-252a, miR-252a-3p and miR-92 were significantly over-expressed during deep aestivation compared with non-aestivation animals. Preliminary analyses of their putative target genes and GO analysis suggest that these miRNAs could play important roles in global transcriptional depression and cell differentiation during aestivation. High-throughput sequencing data and microarray data have been submitted to GEO database.
Growth is a priority trait from the point of view of genetic improvement. Molecular markers linked to quantitative trait loci (QTL) have been regarded as useful for marker-assisted selection (MAS) in complex traits as growth. Using an intermediate F2 cross of slow and fast growth parents, a genetic linkage map of Pacific whiteleg shrimp, Litopenaeusvannamei, based on amplified fragment length polymorphisms (AFLP) and simple sequence repeats (SSR) markers was constructed. Meanwhile, QTL analysis was performed for growth-related traits. The linkage map consisted of 451 marker loci (429 AFLPs and 22 SSRs) which formed 49 linkage groups with an average marker space of 7.6 cM; they spanned a total length of 3627.6 cM, covering 79.50% of estimated genome size. 14 QTLs were identified for growth-related traits, including three QTLs for body weight (BW), total length (TL) and partial carapace length (PCL), two QTLs for body length (BL), one QTL for first abdominal segment depth (FASD), third abdominal segment depth (TASD) and first abdominal segment width (FASW), which explained 2.62 to 61.42% of phenotypic variation. Moreover, comparison of linkage maps between L. vannamei and Penaeusjaponicus was applied, providing a new insight into the genetic base of QTL affecting the growth-related traits. The new results will be useful for conducting MAS breeding schemes in L. vannamei.
The left-right asymmetry of snails, including the direction of shell coiling, is determined by the delayed effect of a maternal gene on the chiral twist that takes place during early embryonic cell divisions. Yet, despite being a well-established classical problem, the identity of the gene and the means by which left-right asymmetry is established in snails remain unknown. We here demonstrate the power of new genomic approaches for identification of the chirality gene, “D”. First, heterozygous (Dd) pond snails Lymnaea stagnalis were self-fertilised or backcrossed, and the genotype of more than six thousand offspring inferred, either dextral (DD/Dd) or sinistral (dd). Then, twenty of the offspring were used for Restriction-site-Associated DNA Sequencing (RAD-Seq) to identify anonymous molecular markers that are linked to the chirality locus. A local genetic map was constructed by genotyping three flanking markers in over three thousand snails. The three markers lie either side of the chirality locus, with one very tightly linked (<0.1 cM). Finally, bacterial artificial chromosomes (BACs) were isolated that contained the three loci. Fluorescent in situ hybridization (FISH) of pachytene cells showed that the three BACs tightly cluster on the same bivalent chromosome. Fibre-FISH identified a region of greater that ∼0.4 Mb between two BAC clone markers that must contain D. This work therefore establishes the resources for molecular identification of the chirality gene and the variation that underpins sinistral and dextral coiling. More generally, the results also show that combining genomic technologies, such as RAD-Seq and high resolution FISH, is a robust approach for mapping key loci in non-model systems.
Catfish has a male-heterogametic (XY) sex determination system, but genes involved in gonadogenesis, spermatogenesis, testicular determination, and sex determination are poorly understood. As a first step of understanding the transcriptome of the testis, here, we conducted RNA-Seq analysis using high throughput Illumina sequencing.
A total of 269.6 million high quality reads were assembled into 193,462 contigs with a N50 length of 806 bp. Of these contigs, 67,923 contigs had hits to a set of 25,307 unigenes, including 167 unique genes that had not been previously identified in catfish. A meta-analysis of expressed genes in the testis and in the gynogen (double haploid female) allowed the identification of 5,450 genes that are preferentially expressed in the testis, providing a pool of putative male-biased genes. Gene ontology and annotation analysis suggested that many of these male-biased genes were involved in gonadogenesis, spermatogenesis, testicular determination, gametogenesis, gonad differentiation, and possibly sex determination.
We provide the first transcriptome-level analysis of the catfish testis. Our analysis would lay the basis for sequential follow-up studies of genes involved in sex determination and differentiation in catfish.
Sheepgrass [Leymus chinensis (Trin.) Tzvel.] is an important perennial forage grass across the Eurasian Steppe and is known for its adaptability to various environmental conditions. However, insufficient data resources in public databases for sheepgrass limited our understanding of the mechanism of environmental adaptations, gene discovery and molecular marker development.
The transcriptome of sheepgrass was sequenced using Roche 454 pyrosequencing technology. We assembled 952,328 high-quality reads into 87,214 unigenes, including 32,416 contigs and 54,798 singletons. There were 15,450 contigs over 500 bp in length. BLAST searches of our database against Swiss-Prot and NCBI non-redundant protein sequences (nr) databases resulted in the annotation of 54,584 (62.6%) of the unigenes. Gene Ontology (GO) analysis assigned 89,129 GO term annotations for 17,463 unigenes. We identified 11,675 core Poaceae-specific and 12,811 putative sheepgrass-specific unigenes by BLAST searches against all plant genome and transcriptome databases. A total of 2,979 specific freezing-responsive unigenes were found from this RNAseq dataset. We identified 3,818 EST-SSRs in 3,597 unigenes, and some SSRs contained unigenes that were also candidates for freezing-response genes. Characterizations of nucleotide repeats and dominant motifs of SSRs in sheepgrass were also performed. Similarity and phylogenetic analysis indicated that sheepgrass is closely related to barley and wheat.
This research has greatly enriched sheepgrass transcriptome resources. The identified stress-related genes will help us to decipher the genetic basis of the environmental and ecological adaptations of this species and will be used to improve wheat and barley crops through hybridization or genetic transformation. The EST-SSRs reported here will be a valuable resource for future gene-phenotype studies and for the molecular breeding of sheepgrass and other Poaceae species.
Recent advances in RNA sequencing (RNA-Seq) have enabled the discovery of novel transcriptomic variations that are not possible with traditional microarray-based methods. Tissue and cell specific transcriptome changes during pathophysiological stress in disease cases versus controls and in response to therapies are of particular interest to investigators studying cardiometabolic diseases. Thus, knowledge on the relationships between sequencing depth and detection of transcriptomic variation is needed for designing RNA-Seq experiments and for interpreting results of analyses. Using deeply sequenced Illumina HiSeq 2000 101 bp paired-end RNA-Seq data derived from adipose of a healthy individual before and after systemic administration of endotoxin (LPS), we investigated the sequencing depths needed for studies of gene expression and alternative splicing (AS). In order to detect expressed genes and AS events, we found that ∼100 to 150 million (M) filtered reads were needed. However, the requirement on sequencing depth for the detection of LPS modulated differential expression (DE) and differential alternative splicing (DAS) was much higher. To detect 80% of events, ∼300 M filtered reads were needed for DE analysis whereas at least 400 M filtered reads were necessary for detecting DAS. Although the majority of expressed genes and AS events can be detected with modest sequencing depths (∼100 M filtered reads), the estimated gene expression levels and exon/intron inclusion levels were less accurate. We report the first study that evaluates the relationship between RNA-Seq depth and the ability to detect DE and DAS in human adipose. Our results suggest that a much higher sequencing depth is needed to reliably identify DAS events than for DE genes.
Comparative genomics is a powerful tool to transfer genomic information from model species to related non-model species. Channel catfish (Ictalurus punctatus) is the primary aquaculture species in the United States. Its existing genome resources such as genomic sequences generated from next generation sequencing, BAC end sequences (BES), physical maps, linkage maps, and integrated linkage and physical maps using BES-associated markers provide a platform for comparative genomic analysis between catfish and other model teleost fish species. This study aimed to gain understanding of genome organizations and similarities among catfish and several sequenced teleost genomes using linkage group 8 (LG8) as a pilot study.
With existing genome resources, 287 unique genes were identified in LG8. Comparative genome analysis indicated that most of these 287 genes on catfish LG8 are located on two homologous chromosomes of zebrafish, medaka, stickleback, and three chromosomes of green-spotted pufferfish. Large numbers of conserved syntenies were identified. Detailed analysis of the conserved syntenies in relation to chromosome level similarities revealed extensive inter-chromosomal and intra-chromosomal rearrangements during evolution. Of the 287 genes, 35 genes were found to be duplicated in the catfish genome, with the vast majority of the duplications being interchromosomal.
Comparative genome analysis is a powerful tool even in the absence of a well-assembled whole genome sequence. In spite of sequence stacking due to low resolution of the linkage and physical maps, conserved syntenies can be identified although the exact gene order and orientation are unknown at present. Through chromosome-level comparative analysis, homologous chromosomes among teleosts can be identified. Syntenic analysis should facilitate annotation of the catfish genome, which in turn, should facilitate functional inference of genes based on their orthology.
Comparative mapping; Synteny; Genome; Chromosome; Linkage map; Physical map; Catfish; Fish
Gene discovery in the Malaysian giant freshwater prawn (Macrobrachium rosenbergii) has been limited to small scale data collection, despite great interest in various research fields related to the commercial significance of this species. Next generation sequencing technologies that have been developed recently and enabled whole transcriptome sequencing (RNA-seq), have allowed generation of large scale functional genomics data sets in a shorter time than was previously possible. Using this technology, transcriptome sequencing of three tissue types: hepatopancreas, gill and muscle, has been undertaken to generate functional genomics data for M. rosenbergii at a massive scale. De novo assembly of 75-bp paired end Ilumina reads has generated 102,230 unigenes. Sequence homology search and in silico prediction have identified known and novel protein coding candidate genes (∼24%), non-coding RNA, and repetitive elements in the transcriptome. Potential markers consisting of simple sequence repeats associated with known protein coding genes have been successfully identified. Using KEGG pathway enrichment, differentially expressed genes in different tissues were systematically represented. The functions of gill and hepatopancreas in the context of neuroactive regulation, metabolism, reproduction, environmental stress and disease responses are described and support relevant experimental studies conducted previously in M. rosenbergii and other crustaceans. This large scale gene discovery represents the most extensive transcriptome data for freshwater prawn. Comparison with model organisms has paved the path to address the possible conserved biological entities shared between vertebrates and crustaceans. The functional genomics resources generated from this study provide the basis for constructing hypotheses for future molecular research in the freshwater shrimp.
Although a large set of full-length transcripts was recently assembled in catfish, annotation of large gene families, especially those with duplications, is still a great challenge. Most often, complexities in annotation cause mis-identification and thereby much confusion in the scientific literature. As such, detailed phylogenetic analysis and/or orthology analysis are required for annotation of genes involved in gene families. The ATP-binding cassette (ABC) transporter gene superfamily is a large gene family that encodes membrane proteins that transport a diverse set of substrates across membranes, playing important roles in protecting organisms from diverse environment.
In this work, we identified a set of 50 ABC transporters in catfish genome. Phylogenetic analysis allowed their identification and annotation into seven subfamilies, including 9 ABCA genes, 12 ABCB genes, 12 ABCC genes, 5 ABCD genes, 2 ABCE genes, 4 ABCF genes and 6 ABCG genes. Most ABC transporters are conserved among vertebrates, though cases of recent gene duplications and gene losses do exist. Gene duplications in catfish were found for ABCA1, ABCB3, ABCB6, ABCC5, ABCD3, ABCE1, ABCF2 and ABCG2.
The whole set of catfish ABC transporters provide the essential genomic resources for future biochemical, toxicological and physiological studies of ABC drug efflux transporters. The establishment of orthologies should allow functional inferences with the information from model species, though the function of lineage-specific genes can be distinct because of specific living environment with different selection pressure.
The insect predator, Arma chinensis, is capable of effectively controlling many pests, such as Colorado potato beetle, cotton bollworm, and mirid bugs. Our previous study demonstrated several life history parameters were diminished for A. chinensis reared on an artificial diet compared to a natural food source like the Chinese oak silk moth pupae. The molecular mechanisms underlying the nutritive impact of the artificial diet on A. chinensis health are unclear. So we utilized transcriptome information to better understand the impact of the artificial diet on A. chinensis at the molecular level.
Illumina HiSeq2000 was used to sequence 4.79 and 4.70 Gb of the transcriptome from pupae-fed and artificial diet-fed A. chinensis libraries, respectively, and a de novo transcriptome assembly was performed (Trinity short read assembler). This resulted in 112,029 and 98,724 contigs, clustered into 54,083 and 54,169 unigenes for pupae-fed and diet-fed A. chinensis, respectively. Unigenes from each sample’s assembly underwent sequence splicing and redundancy removal to acquire non-redundant unigenes. We obtained 55,189 unigenes of A. chinensis, including 12,046 distinct clusters and 43,143 distinct singletons. Unigene sequences were aligned by BLASTx to nr, Swiss-Prot, KEGG and COG (E-value <10−5), and further aligned by BLASTn to nt (E-value <10−5), retrieving proteins of highest sequence similarity with the given unigenes along with their protein functional annotations. Totally, 22,964, 7,898, 18,069, 15,416, 8,066 and 5,341 unigenes were annotated in nr, nt, Swiss-Prot, KEGG, COG and GO, respectively. We compared gene expression variations and found thousands of genes were differentially expressed between pupae-fed and diet-fed A. chinensis.
Our study provides abundant genomic data and offers comprehensive sequence information for studying A. chinensis. Additionally, the physiological roles of the differentially expressed genes enable us to predict effects of some dietary ingredients and subsequently propose formulation improvements to artificial diets.
Genetic diversity is essential for persistence of animal populations over both the short- and long-term. Previous studies suggest that genetic diversity may decrease with population decline due to genetic drift or inbreeding of small populations. For oscillating populations, there are some studies on the relationship between population density and genetic diversity, but these studies were based on short-term observation or in low-density phases. Evidence from rapidly expanding populations is lacking. In this study, genetic diversity of a rapidly expanding population of the Greater long-tailed hamsters during 1984–1990, in the Raoyang County of the North China Plain was studied using DNA microsatellite markers. Results show that genetic diversity was positively correlated with population density (as measured by % trap success), and the increase in population density was correlated with a decrease of genetic differentiation between the sub-population A and B. The genetic diversity tended to be higher in spring than in autumn. Variation in population density and genetic diversity are consistent between sub-population A and B. Such results suggest that dispersal is density- and season-dependent in a rapidly expanding population of the Greater long-tailed hamster. For typically solitary species, increasing population density can increase intra-specific attack, which is a driving force for dispersal. This situation is counterbalanced by decreasing population density caused by genetic drift or inbreeding as the result of small population size. Season is a major factor influencing population density and genetic diversity. Meanwhile, roads, used to be considered as geographical isolation, have less effect on genetic differentiation in a rapidly expanding population. Evidences suggest that gene flow (Nm) is positively correlated with population density, and it is significant higher in spring than that in autumn.