Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer’s disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4′-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4′-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.
Theobroma cacao L. cultivar Matina 1-6 belongs to the most cultivated cacao type. The availability of its genome sequence and methods for identifying genes responsible for important cacao traits will aid cacao researchers and breeders.
We describe the sequencing and assembly of the genome of Theobroma cacao L. cultivar Matina
1-6. The genome of the Matina 1-6 cultivar is 445 Mbp, which is significantly larger than a sequenced Criollo cultivar, and more typical of other cultivars. The chromosome-scale assembly, version 1.1, contains 711 scaffolds covering 346.0 Mbp, with a contig N50 of 84.4 kbp, a scaffold N50 of 34.4 Mbp, and an evidence-based gene set of 29,408 loci. Version 1.1 has 10x the scaffold N50 and 4x the contig N50 as Criollo, and includes 111 Mb more anchored sequence. The version 1.1 assembly has 4.4% gap sequence, while Criollo has 10.9%. Through a combination of haplotype, association mapping and gene expression analyses, we leverage this robust reference genome to identify a promising candidate gene responsible for pod color variation. We demonstrate that green/red pod color in cacao is likely regulated by the R2R3 MYB transcription factor TcMYB113, homologs of which determine pigmentation in Rosaceae, Solanaceae, and Brassicaceae. One SNP within the target site for a highly conserved trans-acting siRNA in dicots, found within TcMYB113, seems to affect transcript levels of this gene and therefore pod color variation.
We report a high-quality sequence and annotation of Theobroma cacao L. and demonstrate its utility in identifying candidate genes regulating traits.
Theobroma cacao L.; genome; Matina 1-6; haplotype phasing; genetic mapping; pod color; MYB113
Genome-wide association study (GWAS) has revolutionized the search for the genetic basis of complex traits. To date, GWAS have generally relied on relatively sparse sampling of nucleotide diversity, which is likely to bias results by preferentially sampling high-frequency SNPs not in complete linkage disequilibrium (LD) with causative SNPs. To avoid these limitations we conducted GWAS with >6 million SNPs identified by sequencing the genomes of 226 accessions of the model legume Medicago truncatula. We used these data to identify candidate genes and the genetic architecture underlying phenotypic variation in plant height, trichome density, flowering time, and nodulation. The characteristics of candidate SNPs differed among traits, with candidates for flowering time and trichome density in distinct clusters of high linkage disequilibrium (LD) and the minor allele frequencies (MAF) of candidates underlying variation in flowering time and height significantly greater than MAF of candidates underlying variation in other traits. Candidate SNPs tagged several characterized genes including nodulation related genes SERK2, MtnodGRP3, MtMMPL1, NFP, CaML3, MtnodGRP3A and flowering time gene MtFD as well as uncharacterized genes that become candidates for further molecular characterization. By comparing sequence-based candidates to candidates identified by in silico 250K SNP arrays, we provide an empirical example of how reliance on even high-density reduced representation genomic makers can bias GWAS results. Depending on the trait, only 30–70% of the top 20 in silico array candidates were within 1 kb of sequence-based candidates. Moreover, the sequence-based candidates tagged by array candidates were heavily biased towards common variants; these comparisons underscore the need for caution when interpreting results from GWAS conducted with sparsely covered genomes.
The sinorhizobia are amongst the most well studied members of nitrogen-fixing root nodule bacteria and contribute substantial amounts of fixed nitrogen to the biosphere. While the alfalfa symbiont Sinorhizobium meliloti RM 1021 was one of the first rhizobial strains to be completely sequenced, little information is available about the genomes of this large and diverse species group.
Here we report the draft assembly and annotation of 48 strains of Sinorhizobium comprising five genospecies. While S. meliloti and S. medicae are taxonomically related, they displayed different nodulation patterns on diverse Medicago host plants, and have differences in gene content, including those involved in conjugation and organic sulfur utilization. Genes involved in Nod factor and polysaccharide biosynthesis, denitrification and type III, IV, and VI secretion systems also vary within and between species. Symbiotic phenotyping and mutational analyses indicated that some type IV secretion genes are symbiosis-related and involved in nitrogen fixation efficiency. Moreover, there is a correlation between the presence of type IV secretion systems, heme biosynthesis and microaerobic denitrification genes, and symbiotic efficiency.
Our results suggest that each Sinorhizobium strain uses a slightly different strategy to obtain maximum compatibility with a host plant. This large genome data set provides useful information to better understand the functional features of five Sinorhizobium species, especially compatibility in legume-Sinorhizobium interactions. The diversity of genes present in the accessory genomes of members of this genus indicates that each bacterium has adopted slightly different strategies to interact with diverse plant genera and soil environments.
The advent of next-generation DNA sequencing (NGS) technologies has led to the development of rapid genome-wide Single Nucleotide Polymorphism (SNP) detection applications in various plant species. Recent improvements in sequencing throughput combined with an overall decrease in costs per gigabase of sequence is allowing NGS to be applied to not only the evaluation of small subsets of parental inbred lines, but also the mapping and characterization of traits of interest in much larger populations. Such an approach, where sequences are used simultaneously to detect and score SNPs, therefore bypassing the entire marker assay development stage, is known as genotyping-by-sequencing (GBS). This review will summarize the current state of GBS in plants and the promises it holds as a genome-wide genotyping application.
genotyping; genotyping-by-sequencing; sequencing; next-generation sequencing; genome-wide association; DNA; SNPs; NGS; GBS
Alfalfa, a perennial, outcrossing species, is a widely planted forage legume producing highly nutritious biomass. Currently, improvement of cultivated alfalfa mainly relies on recurrent phenotypic selection. Marker assisted breeding strategies can enhance alfalfa improvement efforts, particularly if many genome-wide markers are available. Transcriptome sequencing enables efficient high-throughput discovery of single nucleotide polymorphism (SNP) markers for a complex polyploid species.
The transcriptomes of 27 alfalfa genotypes, including elite breeding genotypes, parents of mapping populations, and unimproved wild genotypes, were sequenced using an Illumina Genome Analyzer IIx. De novo assembly of quality-filtered 72-bp reads generated 25,183 contigs with a total length of 26.8 Mbp and an average length of 1,065 bp, with an average read depth of 55.9-fold for each genotype. Overall, 21,954 (87.2%) of the 25,183 contigs represented 14,878 unique protein accessions. Gene ontology (GO) analysis suggested that a broad diversity of genes was represented in the resulting sequences. The realignment of individual reads to the contigs enabled the detection of 872,384 SNPs and 31,760 InDels. High resolution melting (HRM) analysis was used to validate 91% of 192 putative SNPs identified by sequencing. Both allelic variants at about 95% of SNP sites identified among five wild, unimproved genotypes are still present in cultivated alfalfa, and all four US breeding programs also contain a high proportion of these SNPs. Thus, little evidence exists among this dataset for loss of significant DNA sequence diversity from either domestication or breeding of alfalfa. Structure analysis indicated that individuals from the subspecies falcata, the diploid subspecies caerulea, and the tetraploid subspecies sativa (cultivated tetraploid alfalfa) were clearly separated.
We used transcriptome sequencing to discover large numbers of SNPs segregating in elite breeding populations of alfalfa. Little loss of SNP diversity was evident between unimproved and elite alfalfa germplasm. The EST and SNP markers generated from this study are publicly available at the Legume Information System (
http://medsa.comparative-legumes.org/) and can contribute to future alfalfa research and breeding applications.
Single-nucleotide polymorphisms (SNPs, >2000) were discovered by using RNA-seq and allele-specific sequencing approaches in pigeonpea (Cajanus cajan). For making the SNP genotyping cost-effective, successful competitive allele-specific polymerase chain reaction (KASPar) assays were developed for 1616 SNPs and referred to as PKAMs (pigeonpea KASPar assay markers). Screening of PKAMs on 24 genotypes [23 from cultivated species and 1 wild species (Cajanus scarabaeoides)] defined a set of 1154 polymorphic markers (77.4%) with a polymorphism information content (PIC) value from 0.04 to 0.38. One thousand and ninety-four PKAMs showed polymorphisms between parental lines of the reference mapping population (C. cajan ICP 28 × C. scarabaeoides ICPW 94). By using high-quality marker genotyping data on 167 F2 lines from the population, a comprehensive genetic map comprising 875 PKAMs with an average inter-marker distance of 1.11 cM was developed. Previously mapped 35 simple sequence repeat markers were integrated into the PKAM map and an integrated genetic map of 996.21 cM was constructed. Mapped PKAMs showed a higher degree of synteny with the genome of Glycine max followed by Medicago truncatula and Lotus japonicus and least with Vigna unguiculata. These PKAMs will be useful for genetics research and breeding applications in pigeonpea and for utilizing genome information from other legume species.
pigeonpea; SNP; linkage map; comparative genomics; molecular breeding
Polyclonal serum consists of vast collections of antibodies, products of differentiated B-cells. The spectrum of antibody specificities is dynamic and varies with age, physiology, and exposure to pathological insults. The complete repertoire of antibody specificities in blood, the IgOme, is therefore an extraordinarily rich source of information–a molecular record of previous encounters as well as a status report of current immune activity. The ability to profile antibody specificities of polyclonal serum at exceptionally high resolution has been an important and serious challenge which can now be overcome.
Here we illustrate the application of Deep Panning, a method that combines the flexibility of combinatorial phage display of random peptides with the power of high-throughput deep sequencing. Deep Panning is first applied to evaluate the quality and diversity of naïve random peptide libraries. The production of very large data sets, hundreds of thousands of peptides, has revealed unexpected properties of combinatorial random peptide libraries and indicates correctives to ensure the quality of the libraries generated. Next, Deep Panning is used to analyze a model monoclonal antibody in addition to allowing one to follow the dynamics of biopanning and peptide selection. Finally Deep Panning is applied to profile polyclonal sera derived from HIV infected individuals.
The ability to generate and characterize hundreds of thousands of affinity-selected peptides creates an effective means towards the interrogation of the IgOme and understanding of the humoral response to disease. Deep Panning should open the door to new possibilities for serological diagnostics, vaccine design and the discovery of the correlates of immunity to emerging infectious agents.
Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation 1. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Mya). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species 2. Medicago truncatula (Mt) is a long-established model for the study of legume biology. Here we describe the draft sequence of the Mt euchromatin based on a recently completed BAC-assembly supplemented with Illumina-shotgun sequence, together capturing ~94% of all Mt genes. A whole-genome duplication (WGD) approximately 58 Mya played a major role in shaping the Mt genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the Mt genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max (Gm) and Lotus japonicus (Lj). Mt is a close relative of alfalfa (M. sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the Mt genome sequence provides significant opportunities to expand alfalfa’s genomic toolbox.
A comprehensive transcriptome assembly for pigeonpea has been developed by analyzing 128.9 million short Illumina GA IIx single end reads, 2.19 million single end FLX/454 reads, and 18 353 Sanger expressed sequenced tags from more than 16 genotypes. The resultant transcriptome assembly, referred to as CcTA v2, comprised 21 434 transcript assembly contigs (TACs) with an N50 of 1510 bp, the largest one being ∼8 kb. Of the 21 434 TACs, 16 622 (77.5%) could be mapped on to the soybean genome build 1.0.9 under fairly stringent alignment parameters. Based on knowledge of intron junctions, 10 009 primer pairs were designed from 5033 TACs for amplifying intron spanning regions (ISRs). By using in silico mapping of BAC-end-derived SSR loci of pigeonpea on the soybean genome as a reference, putative mapping positions at the chromosome level were predicted for 6284 ISR markers, covering all 11 pigeonpea chromosomes. A subset of 128 ISR markers were analyzed on a set of eight genotypes. While 116 markers were validated, 70 markers showed one to three alleles, with an average of 0.16 polymorphism information content (PIC) value. In summary, the CcTA v2 transcript assembly and ISR markers will serve as a useful resource to accelerate genetic research and breeding applications in pigeonpea.
Cajanus cajan (L.); second-generation sequencing; transcriptome assembly; intron spanning region (ISR) markers
This study reports generation of large-scale genomic resources for pigeonpea, a so-called ‘orphan crop species’ of the semi-arid tropic regions. FLX/454 sequencing carried out on a normalized cDNA pool prepared from 31 tissues produced 494 353 short transcript reads (STRs). Cluster analysis of these STRs, together with 10 817 Sanger ESTs, resulted in a pigeonpea trancriptome assembly (CcTA) comprising of 127 754 tentative unique sequences (TUSs). Functional analysis of these TUSs highlights several active pathways and processes in the sampled tissues. Comparison of the CcTA with the soybean genome showed similarity to 10 857 and 16 367 soybean gene models (depending on alignment methods). Additionally, Illumina 1G sequencing was performed on Fusarium wilt (FW)- and sterility mosaic disease (SMD)-challenged root tissues of 10 resistant and susceptible genotypes. More than 160 million sequence tags were used to identify FW- and SMD-responsive genes. Sequence analysis of CcTA and the Illumina tags identified a large new set of markers for use in genetics and breeding, including 8137 simple sequence repeats, 12 141 single-nucleotide polymorphisms and 5845 intron-spanning regions. Genomic resources developed in this study should be useful for basic and applied research, not only for pigeonpea improvement but also for other related, agronomically important legumes.
Cajanus cajan L.; next generation sequencing; transcriptome assembly; molecular markers and gene discovery
Pigeonpea [Cajanus cajan (L.) Millsp.] is an important legume crop of rainfed agriculture. Despite of concerted research efforts directed to pigeonpea improvement, stagnated productivity of pigeonpea during last several decades may be accounted to prevalence of various biotic and abiotic constraints and the situation is exacerbated by availability of inadequate genomic resources to undertake any molecular breeding programme for accelerated crop improvement. With the objective of enhancing genomic resources for pigeonpea, this study reports for the first time, large scale development of SSR markers from BAC-end sequences and their subsequent use for genetic mapping and hybridity testing in pigeonpea.
A set of 88,860 BAC (bacterial artificial chromosome)-end sequences (BESs) were generated after constructing two BAC libraries by using HindIII (34,560 clones) and BamHI (34,560 clones) restriction enzymes. Clustering based on sequence identity of BESs yielded a set of >52K non-redundant sequences, comprising 35 Mbp or >4% of the pigeonpea genome. These sequences were analyzed to develop annotation lists and subdivide the BESs into genome fractions (e.g., genes, retroelements, transpons and non-annotated sequences). Parallel analysis of BESs for microsatellites or simple sequence repeats (SSRs) identified 18,149 SSRs, from which a set of 6,212 SSRs were selected for further analysis. A total of 3,072 novel SSR primer pairs were synthesized and tested for length polymorphism on a set of 22 parental genotypes of 13 mapping populations segregating for traits of interest. In total, we identified 842 polymorphic SSR markers that will have utility in pigeonpea improvement. Based on these markers, the first SSR-based genetic map comprising of 239 loci was developed for this previously uncharacterized genome. Utility of developed SSR markers was also demonstrated by identifying a set of 42 markers each for two hybrids (ICPH 2671 and ICPH 2438) for genetic purity assessment in commercial hybrid breeding programme.
In summary, while BAC libraries and BESs should be useful for genomics studies, BES-SSR markers, and the genetic map should be very useful for linking the genetic map with a future physical map as well as for molecular breeding in pigeonpea.
Meiosis is a critical process in the reproduction and life cycle of flowering plants in which homologous chromosomes pair, synapse, recombine and segregate. Understanding meiosis will not only advance our knowledge of the mechanisms of genetic recombination, but also has substantial applications in crop improvement. Despite the tremendous progress in the past decade in other model organisms (e.g., Saccharomyces cerevisiae and Drosophila melanogaster), the global identification of meiotic genes in flowering plants has remained a challenge due to the lack of efficient methods to collect pure meiocytes for analyzing the temporal and spatial gene expression patterns during meiosis, and for the sensitive identification and quantitation of novel genes.
A high-throughput approach to identify meiosis-specific genes by combining isolated meiocytes, RNA-Seq, bioinformatic and statistical analysis pipelines was developed. By analyzing the studied genes that have a meiosis function, a pipeline for identifying meiosis-specific genes has been defined. More than 1,000 genes that are specifically or preferentially expressed in meiocytes have been identified as candidate meiosis-specific genes. A group of 55 genes that have mitochondrial genome origins and a significant number of transposable element (TE) genes (1,036) were also found to have up-regulated expression levels in meiocytes.
These findings advance our understanding of meiotic genes, gene expression and regulation, especially the transcript profiles of MGI genes and TE genes, and provide a framework for functional analysis of genes in meiosis.
Next generation sequencing is transforming our understanding of transcriptomes. It can determine the expression level of transcripts with a dynamic range of over six orders of magnitude from multiple tissues, developmental stages or conditions. Patterns of gene expression provide insight into functions of genes with unknown annotation.
The RNA Seq-Atlas presented here provides a record of high-resolution gene expression in a set of fourteen diverse tissues. Hierarchical clustering of transcriptional profiles for these tissues suggests three clades with similar profiles: aerial, underground and seed tissues. We also investigate the relationship between gene structure and gene expression and find a correlation between gene length and expression. Additionally, we find dramatic tissue-specific gene expression of both the most highly-expressed genes and the genes specific to legumes in seed development and nodule tissues. Analysis of the gene expression profiles of over 2,000 genes with preferential gene expression in seed suggests there are more than 177 genes with functional roles that are involved in the economically important seed filling process. Finally, the Seq-atlas also provides a means of evaluating existing gene model annotations for the Glycine max genome.
This RNA-Seq atlas extends the analyses of previous gene expression atlases performed using Affymetrix GeneChip technology and provides an example of new methods to accommodate the increase in transcriptome data obtained from next generation sequencing. Data contained within this RNA-Seq atlas of Glycine max can be explored at http://www.soybase.org/soyseq.
Several lines of evidence indicate that polyploidy occurred by around 54 million years ago, early in the history of legume evolution, but it has not been known whether this event was confined to the papilionoid subfamily (Papilionoideae; e.g. beans, medics, lupins) or occurred earlier. Determining the timing of the polyploidy event is important for understanding whether polyploidy might have contributed to rapid diversification and radiation of the legumes near the origin of the family; and whether polyploidy might have provided genetic material that enabled the evolution of a novel organ, the nitrogen-fixing nodule. Although symbioses with nitrogen-fixing partners have evolved in several lineages in the rosid I clade, nodules are widespread only in legume taxa, being nearly universal in the papilionoids and in the mimosoid subfamily (e.g., mimosas, acacias) – which diverged from the papilionoid legumes around 58 million years ago, soon after the origin of the legumes.
Using transcriptome sequence data from Chamaecrista fasciculata, a nodulating member of the mimosoid clade, we tested whether this species underwent polyploidy within the timeframe of legume diversification. Analysis of gene family branching orders and synonymous-site divergence data from C. fasciculata, Glycine max (soybean), Medicago truncatula, and Vitis vinifera (grape; an outgroup to the rosid taxa) establish that the polyploidy event known from soybean and Medicago occurred after the separation of the mimosoid and papilionoid clades, and at or shortly before the Papilionoideae radiation.
The ancestral legume genome was not fundamentally polyploid. Moreover, because there has not been an independent instance of polyploidy in the Chamaecrista lineage there is no necessary connection between polyploidy and nodulation in legumes. Chamaecrista may serve as a useful model in the legumes that lacks a paleopolyploid history, at least relative to the widely studied papilionoid models.
Scientific data integration and computational service discovery are challenges for the bioinformatic community. This process is made more difficult by the separate and independent construction of biological databases, which makes the exchange of data between information resources difficult and labor intensive. A recently described semantic web protocol, the Simple Semantic Web Architecture and Protocol (SSWAP; pronounced "swap") offers the ability to describe data and services in a semantically meaningful way. We report how three major information resources (Gramene, SoyBase and the Legume Information System [LIS]) used SSWAP to semantically describe selected data and web services.
We selected high-priority Quantitative Trait Locus (QTL), genomic mapping, trait, phenotypic, and sequence data and associated services such as BLAST for publication, data retrieval, and service invocation via semantic web services. Data and services were mapped to concepts and categories as implemented in legacy and de novo community ontologies. We used SSWAP to express these offerings in OWL Web Ontology Language (OWL), Resource Description Framework (RDF) and eXtensible Markup Language (XML) documents, which are appropriate for their semantic discovery and retrieval. We implemented SSWAP services to respond to web queries and return data. These services are registered with the SSWAP Discovery Server and are available for semantic discovery at http://sswap.info.
A total of ten services delivering QTL information from Gramene were created. From SoyBase, we created six services delivering information about soybean QTLs, and seven services delivering genetic locus information. For LIS we constructed three services, two of which allow the retrieval of DNA and RNA FASTA sequences with the third service providing nucleic acid sequence comparison capability (BLAST).
The need for semantic integration technologies has preceded available solutions. We report the feasibility of mapping high priority data from local, independent, idiosyncratic data schemas to common shared concepts as implemented in web-accessible ontologies. These mappings are then amenable for use in semantic web services. Our implementation of approximately two dozen services means that biological data at three large information resources (Gramene, SoyBase, and LIS) is available for programmatic access, semantic searching, and enhanced interaction between the separate missions of these resources.
The nutritional and economic value of many crops is effectively a function of seed protein and oil content. Insight into the genetic and molecular control mechanisms involved in the deposition of these constituents in the developing seed is needed to guide crop improvement. A quantitative trait locus (QTL) on Linkage Group I (LG I) of soybean (Glycine max (L.) Merrill) has a striking effect on seed protein content.
A soybean near-isogenic line (NIL) pair contrasting in seed protein and differing in an introgressed genomic segment containing the LG I protein QTL was used as a resource to demarcate the QTL region and to study variation in transcript abundance in developing seed. The LG I QTL region was delineated to less than 8.4 Mbp of genomic sequence on chromosome 20. Using Affymetrix® Soy GeneChip and high-throughput Illumina® whole transcriptome sequencing platforms, 13 genes displaying significant seed transcript accumulation differences between NILs were identified that mapped to the 8.4 Mbp LG I protein QTL region.
This study identifies gene candidates at the LG I protein QTL for potential involvement in the regulation of protein content in the soybean seed. The results demonstrate the power of complementary approaches to characterize contrasting NILs and provide genome-wide transcriptome insight towards understanding seed biology and the soybean genome.
High-throughput DNA sequencing has enabled systems biology to begin to address areas in health, agricultural and basic biological research. Concomitant with the opportunities is an absolute necessity to manage significant volumes of high-dimensional and inter-related data and analysis. Alpheus is an analysis pipeline, database and visualization software for use with massively parallel DNA sequencing technologies that feature multi-gigabase throughput characterized by relatively short reads, such as Illumina-Solexa (sequencing-by-synthesis), Roche-454 (pyrosequencing) and Applied Biosystem’s SOLiD (sequencing-by-ligation). Alpheus enables alignment to reference sequence(s), detection of variants and enumeration of sequence abundance, including expression levels in transcriptome sequence. Alpheus is able to detect several types of variants, including non-synonymous and synonymous single nucleotide polymorphisms (SNPs), insertions/deletions (indels), premature stop codons, and splice isoforms. Variant detection is aided by the ability to filter variant calls based on consistency, expected allele frequency, sequence quality, coverage, and variant type in order to minimize false positives while maximizing the identification of true positives. Alpheus also enables comparisons of genes with variants between cases and controls or bulk segregant pools. Sequence-based differential expression comparisons can be developed, with data export to SAS JMP Genomics for statistical analysis.
Alpheus; sequencing-by-synthesis; pyrosequencing; GMAP; GSNAP; resequencing; transcriptome sequencing
The Soybean Consensus Map 4.0 facilitated the anchoring of 95.6% of the soybean whole genome sequence developed by the Joint Genome Institute, Department of Energy, but its marker density was only sufficient to properly orient 66% of the sequence scaffolds. The discovery and genetic mapping of more single nucleotide polymorphism (SNP) markers were needed to anchor and orient the remaining genome sequence. To that end, next generation sequencing and high-throughput genotyping were combined to obtain a much higher resolution genetic map that could be used to anchor and orient most of the remaining sequence and to help validate the integrity of the existing scaffold builds.
A total of 7,108 to 25,047 predicted SNPs were discovered using a reduced representation library that was subsequently sequenced by the Illumina sequence-by-synthesis method on the clonal single molecule array platform. Using multiple SNP prediction methods, the validation rate of these SNPs ranged from 79% to 92.5%. A high resolution genetic map using 444 recombinant inbred lines was created with 1,790 SNP markers. Of the 1,790 mapped SNP markers, 1,240 markers had been selectively chosen to target existing unanchored or un-oriented sequence scaffolds, thereby increasing the amount of anchored sequence to 97%.
We have demonstrated how next generation sequencing was combined with high-throughput SNP detection assays to quickly discover large numbers of SNPs. Those SNPs were then used to create a high resolution genetic map that assisted in the assembly of scaffolds from the 8× whole genome shotgun sequences into pseudomolecules corresponding to chromosomes of the organism.
Schizophrenia (SCZ) is a common, disabling mental illness with high heritability but complex, poorly understood genetic etiology. As the first phase of a genomic convergence analysis of SCZ, we generated 16.7 billion nucleotides of short read, shotgun sequences of cDNA from post-mortem cerebellar cortices of 14 patients and six, matched controls. A rigorous analysis pipeline was developed for analysis of digital gene expression studies. Sequences aligned to approximately 33,200 transcripts in each sample, with average coverage of 450 reads per gene. Following adjustments for confounding clinical, sample and experimental sources of variation, 215 genes differed significantly in expression between cases and controls. Golgi apparatus, vesicular transport, membrane association, Zinc binding and regulation of transcription were over-represented among differentially expressed genes. Twenty three genes with altered expression and involvement in presynaptic vesicular transport, Golgi function and GABAergic neurotransmission define a unifying molecular hypothesis for dysfunction in cerebellar cortex in SCZ.
Tall fescue (Festuca arundinacea Schreb) is a major cool season forage and turf grass species grown in the temperate regions of the world. In this paper we report the generation of a tall fescue expressed sequence tag (EST) database developed from nine cDNA libraries representing tissues from different plant organs, developmental stages, and abiotic stress factors. The results of inter-library and library-specific in silico expression analyses of these ESTs are also reported.
A total of 41,516 ESTs were generated from nine cDNA libraries of tall fescue representing tissues from different plant organs, developmental stages, and abiotic stress conditions. The Festuca Gene Index (FaGI) has been established. To date, this represents the first publicly available tall fescue EST database. In silico gene expression studies using these ESTs were performed to understand stress responses in tall fescue. A large number of ESTs of known stress response gene were identified from stressed tissue libraries. These ESTs represent gene homologues of heat-shock and oxidative stress proteins, and various transcription factor protein families. Highly expressed ESTs representing genes of unknown functions were also identified in the stressed tissue libraries.
FaGI provides a useful resource for genomics studies of tall fescue and other closely related forage and turf grass species. Comparative genomic analyses between tall fescue and other grass species, including ryegrasses (Lolium sp.), meadow fescue (F. pratensis) and tetraploid fescue (F. arundinacea var glaucescens) will benefit from this database. These ESTs are an excellent resource for the development of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) PCR-based molecular markers.
Guar, Cyamopsis tetragonoloba (L.) Taub, is a member of the Leguminosae (Fabaceae) family and is economically the most important of the four species in the genus. The endosperm of guar seed is a rich source of mucilage or gum, which forms a viscous gel in cold water, and is used as an emulsifier, thickener and stabilizer in a wide range of foods and industrial applications. Guar gum is a galactomannan, consisting of a linear (1→4)-β-linked D-mannan backbone with single-unit, (1→6)-linked, α-D-galactopyranosyl side chains. To better understand regulation of guar seed development and galactomannan metabolism we created cDNA libraries and a resulting EST dataset from different developmental stages of guar seeds.
A database of 16,476 guar seed ESTs was constructed, with 8,163 and 8,313 ESTs derived from cDNA libraries I and II, respectively. Library I was constructed from seeds at an early developmental stage (15–25 days after flowering, DAF), and library II from seeds at 30–40 DAF. Quite different sets of genes were represented in these two libraries. Approximately 27% of the clones were not similar to known sequences, suggesting that these ESTs represent novel genes or may represent non-coding RNA. The high flux of energy into carbohydrate and storage protein synthesis in guar seeds was reflected by a high representation of genes annotated as involved in signal transduction, carbohydrate metabolism, chaperone and proteolytic processes, and translation and ribosome structure. Guar unigenes involved in galactomannan metabolism were identified. Among the seed storage proteins, the most abundant contig represented a conglutin accounting for 3.7% of the total ESTs from both libraries.
The present EST collection and its annotation provide a resource for understanding guar seed biology and galactomannan metabolism.
In this study, we addressed whether a single 454 Life Science GS20 sequencing run provides new gene discovery from a normalized cDNA library, and whether the short reads produced via this technology are of value in gene structure annotation.
A single 454 GS20 sequencing run on adapter-ligated cDNA, from a normalized cDNA library, generated 292,465 reads that were reduced to 252,384 reads with an average read length of 92 nucleotides after cleaning. After clustering and assembly, a total of 184,599 unique sequences were generated containing over 400 SSRs. The 454 sequences generated hits to more genes than a comparable amount of sequence from MtGI. Although short, the 454 reads are of sufficient length to map to a unique genome location as effectively as longer ESTs produced by conventional sequencing. Functional interpretation of the sequences was carried out by Gene Ontology assignments from matches to Arabidopsis and was shown to cover a broad range of GO categories. 53,796 assemblies and singletons (29%) had no match in the existing MtGI. Within the previously unobserved Medicago transcripts, thousands had matches in a comprehensive protein database and one or more of the TIGR Plant Gene Indices. Approximately 20% of these novel sequences could be found in the Medicago genome sequence. A total of 70,026 reads generated by the 454 technology were mapped to 785 Medicago finished BACs using PASA and over 1,000 gene models required modification. In parallel to 454 sequencing, 4,445 5'-prime reads were generated by conventional sequencing using the same library and from the assembled sequences it was shown to contain about 52% full length cDNAs encoding proteins from 50 to over 500 amino acids in length.
Due to the large number of reads afforded by the 454 DNA sequencing technology, it is effective in revealing the expression of transcripts from a broad range of GO categories and contains many rare transcripts in normalized cDNA libraries, although only a limited portion of their sequence is uncovered. As with longer ESTs, 454 reads can be mapped uniquely onto genomic sequence to provide support for, and modifications of, gene predictions.
The Medicago Genome Initiative (MGI) is a database of EST sequences
of the model legume Medicago truncatula. The database
is available to the public and has resulted from a collaborative
research effort between the Samuel Roberts Noble Foundation and
the National Center for Genome Resources to investigate the genome
of M.truncatula. MGI is part of the greater integrated Medicago functional genomics program at the Noble
.org), which is taking a global approach in studying the
genetic and biochemical events associated with the growth, development
and environmental interactions of this model legume. Our approach
will include: large-scale EST sequencing, gene expression profiling,
the generation of M.truncatula activation-tagged
and promoter trap insertion mutants, high-throughput metabolic profiling,
and proteome studies. These multidisciplinary information pools
will be interfaced with one another to provide scientists with an
integrated, holistic set of tools to address fundamental questions pertaining
to legume biology. The public interface to the MGI database can
be accessed at http://www.ncgr.org/research/mgi.
Chickpea (Cicer arietinum L.) is an important legume crop in the semi-arid regions of Asia and Africa. Gains in crop productivity have been low however, particularly because of biotic and abiotic stresses. To help enhance crop productivity using molecular breeding techniques, next generation sequencing technologies such as Roche/454 and Illumina/Solexa were used to determine the sequence of most gene transcripts and to identify drought-responsive genes and gene-based molecular markers. A total of 103 215 tentative unique sequences (TUSs) have been produced from 435 018 Roche/454 reads and 21 491 Sanger expressed sequence tags (ESTs). Putative functions were determined for 49 437 (47.8%) of the TUSs, and gene ontology assignments were determined for 20 634 (41.7%) of the TUSs. Comparison of the chickpea TUSs with the Medicago truncatula genome assembly (Mt 3.5.1 build) resulted in 42 141 aligned TUSs with putative gene structures (including 39 281 predicted intron/splice junctions). Alignment of ∼37 million Illumina/Solexa tags generated from drought-challenged root tissues of two chickpea genotypes against the TUSs identified 44 639 differentially expressed TUSs. The TUSs were also used to identify a diverse set of markers, including 728 simple sequence repeats (SSRs), 495 single nucleotide polymorphisms (SNPs), 387 conserved orthologous sequence (COS) markers, and 2088 intron-spanning region (ISR) markers. This resource will be useful for basic and applied research for genome analysis and crop improvement in chickpea.
chickpea; next generation sequencing; transcriptome; drought-responsive genes; markers