Current Genome Assembly Research Articles

Genome-wide identification of phenolic acid biosynthetic genes in Salvia miltiorrhiza.

Twenty-nine genes related to phenolic acid biosynthesis were identified in the Salvia miltiorrhiza genome. Nineteen of these are described for the first time, with ten genes experimentally correlating to phenolic acid biosynthesis. Vast stores of secondary metabolites exist in plants, many of which possess biological activities related to human health. Phenolic acid derivatives are a class of valuable bioactive pharmaceuticals abundant in the widely used Chinese medicinal herb, Salvia miltiorrhiza. The biosynthetic pathway for phenolic acids differs in this species from that of other investigated plants. However, the molecular basis for this is unknown, with systematic analysis of the genes involved not yet performed. As the first step towards unraveling this complex biosynthetic pathway in S. miltiorrhiza, the current genome assembly was searched for putatively involved genes. Twenty-nine genes were revealed, 19 of which are described here for the first time. These include 15 genes predicted in the phenylpropanoid pathway; seven genes in the tyrosine-derived pathway; six genes encoding putative hydroxycinnamoyltransferases, and one CYP98A, namely CYP98A78. The promoter regions, gene structures and expression patterns of these genes were examined. Furthermore, conserved domains and phylogenetic relationships with homologous proteins in other species were revealed. Most of the key enzymes, including 4-coumarate: CoA ligase, 4-hydroxyphenylpyruvate reductase and hydroxycinnamoyltransferase, were found in multiple copies, each exhibiting different characteristics. Ten genes putatively involved in rosmarinic acid biosynthesis are also described. These findings provide a foundation for further analysis of this complex and diverse pathway, with potential to enhance the synthesis of water-soluble medicinal compounds in S. miltiorrhiza.

Molecular evolution of the porcine type I interferon family: subtype-specific expression and antiviral activity.

Type I interferons (IFNs), key antiviral cytokines, evolve to adapt with ever-changing viral threats during vertebrate speciation. Due to novel pathogenic pressure associated with Suidae speciation and domestication, porcine IFNs evolutionarily engender both molecular and functional diversification, which have not been well addressed in pigs, an important livestock species and animal model for biomedical sciences. Annotation of current swine genome assembly Sscrofa10.2 reveals 57 functional genes and 16 pseudogenes of type I IFNs. Subfamilies of multiple IFNA, IFNW and porcine-specific IFND genes are separated into four clusters with ∼60 kb intervals within the IFNB/IFNE bordered region in SSC1, and each cluster contains mingled subtypes of IFNA, IFNW and IFND. Further curation of the 57 functional IFN genes indicates that they include 18 potential artifactual duplicates. We performed phylogenetic construction as well as analyses of gene duplication/conversion and natural selection and showed that porcine type I IFN genes have been undergoing active diversification through both gene duplication and conversion. Extensive analyses of the non-coding sequences proximal to all IFN coding regions identified several genomic repetitive elements significantly associated with different IFN subtypes. Family-wide studies further revealed their molecular diversity with respect to differential expression and restrictive activity on the resurgence of a porcine endogenous retrovirus. Based on predicted 3-D structures of representative animal IFNs and inferred activity, we categorized the general functional propensity underlying the structure-activity relationship. Evidence indicates gene expansion of porcine type I IFNs. Genomic repetitive elements that associated with IFN subtypes may serve as molecular signatures of respective IFN subtypes and genomic mechanisms to mediate IFN gene evolution and expression. In summary, the porcine type I IFN profile has been phylogenetically defined family-wide and linked to diverse expression and antiviral activity, which is important information for further biological studies across the porcine type I IFN family.

Polymorphism identification and improved genome annotation of Brassica rapa through Deep RNA sequencing.

The mapping and functional analysis of quantitative traits in Brassica rapa can be greatly improved with the availability of physically positioned, gene-based genetic markers and accurate genome annotation. In this study, deep transcriptome RNA sequencing (RNA-Seq) of Brassica rapa was undertaken with two objectives: SNP detection and improved transcriptome annotation. We performed SNP detection on two varieties that are parents of a mapping population to aid in development of a marker system for this population and subsequent development of high-resolution genetic map. An improved Brassica rapa transcriptome was constructed to detect novel transcripts and to improve the current genome annotation. This is useful for accurate mRNA abundance and detection of expression QTL (eQTLs) in mapping populations. Deep RNA-Seq of two Brassica rapa genotypes—R500 (var. trilocularis, Yellow Sarson) and IMB211 (a rapid cycling variety)—using eight different tissues (root, internode, leaf, petiole, apical meristem, floral meristem, silique, and seedling) grown across three different environments (growth chamber, greenhouse and field) and under two different treatments (simulated sun and simulated shade) generated 2.3 billion high-quality Illumina reads. A total of 330,995 SNPs were identified in transcribed regions between the two genotypes with an average frequency of one SNP in every 200 bases. The deep RNA-Seq reassembled Brassica rapa transcriptome identified 44,239 protein-coding genes. Compared with current gene models of B. rapa, we detected 3537 novel transcripts, 23,754 gene models had structural modifications, and 3655 annotated proteins changed. Gaps in the current genome assembly of B. rapa are highlighted by our identification of 780 unmapped transcripts. All the SNPs, annotations, and predicted transcripts can be viewed at http://phytonetworks.ucdavis.edu/.

Assessment and improvement of Indian-origin rhesus macaque and Mauritian-origin cynomolgus macaque genome annotations using deep transcriptome sequencing data.

BackgroundThe genome annotations of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques, two of the most common non‐human primate animal models, are limited.MethodsWe analyzed large‐scale macaque RNA‐based next‐generation sequencing (RNAseq) data to identify un‐annotated macaque transcripts.ResultsFor both macaque species, we uncovered thousands of novel isoforms for annotated genes and thousands of un‐annotated intergenic transcripts enriched with non‐coding RNAs. We also identified thousands of transcript sequences which are partially or completely ‘missing’ from current macaque genome assemblies. We showed that many newly identified transcripts were differentially expressed during SIV infection of rhesus macaques or during Ebola virus infection of cynomolgus macaques.ConclusionsFor two important macaque species, we uncovered thousands of novel isoforms and un‐annotated intergenic transcripts including coding and non‐coding RNAs, polyadenylated and non‐polyadenylated transcripts. This resource will greatly improve future macaque studies, as demonstrated by their applications in infectious disease studies.

A ddRAD-based genetic map and its integration with the genome assembly of Japanese eel (Anguilla japonica) provides insights into genome evolution after the teleost-specific genome duplication

BackgroundRecent advancements in next-generation sequencing technology have enabled cost-effective sequencing of whole or partial genomes, permitting the discovery and characterization of molecular polymorphisms. Double-digest restriction-site associated DNA sequencing (ddRAD-seq) is a powerful and inexpensive approach to developing numerous single nucleotide polymorphism (SNP) markers and constructing a high-density genetic map. To enrich genomic resources for Japanese eel (Anguilla japonica), we constructed a ddRAD-based genetic map using an Ion Torrent Personal Genome Machine and anchored scaffolds of the current genome assembly to 19 linkage groups of the Japanese eel. Furthermore, we compared the Japanese eel genome with genomes of model fishes to infer the history of genome evolution after the teleost-specific genome duplication.ResultsWe generated the ddRAD-based linkage map of the Japanese eel, where the maps for female and male spanned 1748.8 cM and 1294.5 cM, respectively, and were arranged into 19 linkage groups. A total of 2,672 SNP markers and 115 Simple Sequence Repeat markers provide anchor points to 1,252 scaffolds covering 151 Mb (13%) of the current genome assembly of the Japanese eel. Comparisons among the Japanese eel, medaka, zebrafish and spotted gar genomes showed highly conserved synteny among teleosts and revealed part of the eight major chromosomal rearrangement events that occurred soon after the teleost-specific genome duplication.ConclusionsThe ddRAD-seq approach combined with the Ion Torrent Personal Genome Machine sequencing allowed us to conduct efficient and flexible SNP genotyping. The integration of the genetic map and the assembled sequence provides a valuable resource for fine mapping and positional cloning of quantitative trait loci associated with economically important traits and for investigating comparative genomics of the Japanese eel.

Acute Injection and Chronic Perfusion of Kisspeptin Elicit Gonadotropins Release but Fail to Trigger Ovulation in the Mare1

Kisspeptin has emerged as the most potent gonadotropin-releasing hormone (GnRH) secretagogue and appears to represent the penultimate step in the central control of reproduction. In the sheep, we showed that kisspeptin could be used to manipulate gonadotropin secretion and control ovulation. Prompted by these results, we decided to investigate whether kisspeptin could be used as an ovulation-inducing agent in another photoperiodic domestic mammal, the horse. Equine kisspeptin-10 (eKp10) was administered intravenously as bolus injections or short- to long-term perfusions to Welsh pony mares, either during the anestrus season or at various stages of the cycle during the breeding season. In all the experimental conditions, eKp10 reliably increased peripheral concentrations of both luteinizing hormone and follicle-stimulating hormone. The nature of the response to eKp10 was consistent across experimental conditions and physiological states: the increase in gonadotropins was always rapid and essentially transient even when eKp10 was perfused for prolonged periods. Furthermore, eKp10 consistently failed to induce ovulation in the mare. To gain insights into the underlying mechanisms, we used acute injections or perfusions of GnRH. We also cloned the equine orthologues of the kisspeptin precursor and Kiss1r; this was justified by the facts that the current equine genome assembly predicted an amino acid difference between eKp10 and Kp10 in other species while an equine orthologue for Kiss1r was missing altogether. In light of these findings, potential reasons for the divergence in the response to kisspeptin between ewe and mare are discussed. Our data highlight that kisspeptin is not a universal ovulation-inducing agent.

Water buffalo genome characterization by the Illumina BovineHD BeadChip.

To define the best strategies for genomic association studies and genomic selection, it is necessary to determine the extent of linkage disequilibrium (LD) and the genetic structure of the study population. The current study evaluated the transference of genomic information contained in the Illumina BovineHD BeadChip from cattle to buffaloes, and assessed the extent of the LD in buffaloes. Of the 688,593 bovine single nucleotide polymorphism (SNP) that were successfully genotyped from the 384 buffalo samples, only 16,580 markers were polymorphic, and had minor allele frequencies greater than 0.05. A total of 16,580 polymorphic SNPs were identified, which were uniformly distributed throughout the autosomes, because the density and mean distance between markers were similar for all autosomes. The average minor allele frequency for the 16,580 SNPs was 0.23. The overall mean LD for pairs of adjacent markers was 0.29 and 0.71, when measured as for r2 and |D'|, respectively. The 16,580 polymorphic SNPs were matched to Bos taurus chromosome in the current bovine genome assembly (Btau 4.2), and could be utilized in association studies. In conclusion, the Illumina BovineHD BeadChip contains approximately 16,580 polymorphic markers for the water buffalo, which are broadly distributed across the genome. These data could be used in genomic association and genomic selection studies; however, it might be necessary to develop a panel with specific SNP markers for water buffaloes.

The α-gliadin genes from Brachypodium distachyon L. provide evidence for a significant gap in the current genome assembly

Brachypodium distachyon, is a new model plant for most cereal crops while gliadin is a class of wheat storage proteins related with wheat quality attributes. In the published B. distachyon genome sequence databases, no gliadin gene is found. In the current study, a number of gliadin genes in B. distachyon were isolated, which is contradictory to the results of genome sequencing projects. In our study, the B. distachyon seeds were found to have no gliadin protein expression by gel electrophoresis, reversed-phase high-performance liquid chromatography and Western blotting analysis. However, Southern blotting revealed a presence of more than ten copies of α-gliadin coding genes in B. distachyon. By means of AS-PCR amplification, four novel full-ORF α-gliadin genes, and 26 pseudogenes with at least one stop codon as well as their promoter regions were cloned and sequenced from different Brachypodium accessions. Sequence analysis revealed a few of single-nucleotide polymorphisms among these genes. Most pseudogenes were resulted from a C to T change, leading to the generation of TAG or TAA in-frame stop codon. To compare both the full-ORFs and the pseudogenes among Triticum and Triticum-related species, their structural characteristics were analyzed. Based on the four T cell stimulatory toxic epitopes and two ployglutamine domains, Aegilops, Triticum, and Brachypodium species were found to be more closely related. The phylogenetic analysis further revealed that B. distachyon was more closely related to Aegilops tauschii, Aegilops umbellulata, and the A or D genome of Triticum aestivum. The α-gliadin genes were able to express successfully in E. coli using the functional T7 promoter. The relative and absolute quantification of the transcripts of α-gliadin genes in wheat was much higher than that in B. distachyon. The abundant pseudogenes may affect the transcriptional and/or posttranscriptional level of the α-gliadin in B. distachyon.

Characterization of the Z lineage Major histocompatability complex class I genes in zebrafish

Zebrafish (Danio rerio) are a valuable model for studying immunity, infection, and hematopoietic disease and have recently been employed for transplantation assays. However, the lack of syngeneic zebrafish creates challenges with identifying immune-matched individuals. The MHC class I genes, which mediate allogeneic recognition in mammals, have been grouped into three broad lineages in zebrafish: the classical U genes on chromosome 19, the Z genes which have been reported to map to chromosome 1, and the L genes that map to multiple loci. Transplantations between individual zebrafish that are matched at the U locus fail to consistently engraft suggesting that additional loci contribute to allogeneic recognition. Although two full-length zebrafish Z transcripts have been described, the genomic organization and diversity of these genes have not been reported. Herein we define ten Z genes on chromosomes 1 and 3 and on an unplaced genomic scaffold. We report that neither of the Z transcripts previously described match the current genome assembly and classify these transcripts as additional gene loci. We characterize full-length transcripts for 9 of these 12 genes. We demonstrate a high level of expression variation of the Z genes between individual zebrafish suggestive of haplotypic variation. We report low level sequence variation for individual Z genes between individual zebrafish reflecting a possible nonclassical function, although these molecules may still contribute to allogeneic recognition. Finally, we present a gene nomenclature system for the Z genes consistent with MHC nomenclature in other species and with the zebrafish gene nomenclature guidelines.

Application of Genotyping-by-Sequencing on Semiconductor Sequencing Platforms: A Comparison of Genetic and Reference-Based Marker Ordering in Barley

The rapid development of next-generation sequencing platforms has enabled the use of sequencing for routine genotyping across a range of genetics studies and breeding applications. Genotyping-by-sequencing (GBS), a low-cost, reduced representation sequencing method, is becoming a common approach for whole-genome marker profiling in many species. With quickly developing sequencing technologies, adapting current GBS methodologies to new platforms will leverage these advancements for future studies. To test new semiconductor sequencing platforms for GBS, we genotyped a barley recombinant inbred line (RIL) population. Based on a previous GBS approach, we designed bar code and adapter sets for the Ion Torrent platforms. Four sets of 24-plex libraries were constructed consisting of 94 RILs and the two parents and sequenced on two Ion platforms. In parallel, a 96-plex library of the same RILs was sequenced on the Illumina HiSeq 2000. We applied two different computational pipelines to analyze sequencing data; the reference-independent TASSEL pipeline and a reference-based pipeline using SAMtools. Sequence contigs positioned on the integrated physical and genetic map were used for read mapping and variant calling. We found high agreement in genotype calls between the different platforms and high concordance between genetic and reference-based marker order. There was, however, paucity in the number of SNP that were jointly discovered by the different pipelines indicating a strong effect of alignment and filtering parameters on SNP discovery. We show the utility of the current barley genome assembly as a framework for developing very low-cost genetic maps, facilitating high resolution genetic mapping and negating the need for developing de novo genetic maps for future studies in barley. Through demonstration of GBS on semiconductor sequencing platforms, we conclude that the GBS approach is amenable to a range of platforms and can easily be modified as new sequencing technologies, analysis tools and genomic resources develop.

Analysis of cattle olfactory subgenome: the first detail study on the characteristics of the complete olfactory receptor repertoire of a ruminant

BackgroundMammalian olfactory receptors (ORs) are encoded by the largest mammalian multigene family. Understanding the OR gene repertoire in the cattle genome could lead to link the effects of genetic differences in these genes to variations in olfaction in cattle.ResultsWe report here a whole genome analysis of the olfactory receptor genes of Bos taurus using conserved OR gene-specific motifs and known OR protein sequences from diverse species. Our analysis, using the current cattle genome assembly UMD 3.1 covering 99.9% of the cattle genome, shows that the cattle genome contains 1,071 OR-related sequences including 881 functional, 190 pseudo, and 352 partial OR sequences. The OR genes are located in 49 clusters on 26 cattle chromosomes. We classified them into 18 families consisting of 4 Class I and 14 Class II families and these were further grouped into 272 subfamilies. Comparative analyses of the OR genes of cattle, pigs, humans, mice, and dogs showed that 6.0% (n = 53) of functional OR cattle genes were species-specific. We also showed that significant copy number variations are present in the OR repertoire of the cattle from the analysis of 10 selected OR genes.ConclusionOur analysis revealed the almost complete OR gene repertoire from an individual cattle genome. Though the number of OR genes were lower than in pigs, the analysis of the genetic system of cattle ORs showed close similarities to that of the pig.

The shared genomic architecture of human nucleolar organizer regions

The short arms of the five acrocentric human chromosomes harbor sequences that direct the assembly and function of the nucleolus, one of the key functional domains of the nucleus, yet they are absent from the current human genome assembly. Here we describe the genomic architecture of these human nucleolar organizers. Sequences distal and proximal to ribosomal gene arrays are conserved among the acrocentric chromosomes, suggesting they are sites of frequent recombination. Although previously believed to be heterochromatic, characterization of these two flanking regions reveals that they share a complex genomic architecture similar to other euchromatic regions of the genome, but they have distinct genomic characteristics. Proximal sequences are almost entirely segmentally duplicated, similar to the regions bordering centromeres. In contrast, the distal sequence is predominantly unique to the acrocentric short arms and is dominated by a very large inverted repeat. We show that the distal element is localized to the periphery of the nucleolus, where it appears to anchor the ribosomal gene repeats. This, combined with its complex chromatin structure and transcriptional activity, suggests that this region is involved in nucleolar organization. Our results provide a platform for investigating the role of NORs in nucleolar formation and function, and open the door for determining the role of these regions in the well-known empirical association of nucleoli with pathology.

Insight from the lamprey genome: Glimpsing early vertebrate development via neuroendocrine-associated genes and shared synteny of gonadotropin-releasing hormone (GnRH)

Study of the ancient lineage of jawless vertebrates is key to understanding the origins of vertebrate biology. The establishment of the neuroendocrine system with the hypothalamic–pituitary axis at its crux is of particular interest. Key neuroendocrine hormones in this system include the pivotal gonadotropin-releasing hormones (GnRHs) responsible for controlling reproduction via the pituitary. Previous data incorporating several lines of evidence showed all known vertebrate GnRHs were grouped into four paralogous lineages: GnRH1, 2, 3 and 4; with proposed evolutionary paths. Using the currently available lamprey genome assembly, we searched genes of the neuroendocrine system and summarize here the details representing the state of the current lamprey genome assembly. Additionally, we have analyzed in greater detail the evolutionary history of the GnRHs based on the information of the genomic neighborhood of the paralogs in lamprey as compared to other gnathostomes. Significantly, the current evidence suggests that two genome duplication events (both 1R and 2R) that generated the different fish and tetrapod paralogs took place before the divergence of the ancestral agnathans and gnathostome lineages. Syntenic analysis supports this evidence in that the previously-classified type IV GnRHs in lamprey (lGnRH-I and -III) share a common ancestry with GnRH2 and 3, and thus are no longer considered type IV GnRHs. Given the single amino acid difference between lGnRH-II and GnRH2 we propose that a GnRH2-like gene existed before the lamprey/gnathostome split giving rise to lGnRH-II and GnRH2. Furthermore, paralogous type 3 genes (lGnRH-I/III and GnRH3) evolved divergent structure/function in lamprey and gnathostome lineages.

Exceptionally Long CDR3H Are Not Isotype Restricted in Bovine Immunoglobulins

Exceptionally long third complementarity determining regions of the heavy chain (CDR3H) were previously described as a specificity of bovine IgG and IgM immunoglobulins. In addition, the genomic organization of the immunoglobulin heavy chain locus remains to be elucidated with a special focus on the number of variable segments (IGHV). By analyzing the variable regions according to the isotype-specific PCR using cDNA-PCR, we were able to prove the existence of exceptional long CDR3H in all bovine isotypes. The corresponding sequences of three distinct amplicons were grouped according to the length of the CDR3H. Sequences of CDR3H possessed 5 to 10, 12 to 31 or at least 48 amino acid residues. Long and mid-length CDR3H were composed of mainly hydrophilic amino acid residues, while short CDR3H also contained hydrophobic amino acid residues. All sequences with long CDR3H were related to the germline variable segment 10. Using the current genome assembly, Bos taurus NCBI build 6.1, the genomic organization of the bovine immunoglobulin heavy-chain locus was analyzed. A main locus was investigated on BTA21. Exons coding for variable, diversity, and joining segments, as well as for the constant regions of different isotypes, were also localized on BTA7, BTA8, and BTA20. Together with the information from unplaced contigs, 36 IGHV were detected of which 13 are putatively functional. Phylogenetic analysis revealed two bovine IGHV families (boVH1, boVH2). Thus, the existence of the two bovine families suggested was demonstrated, where boVH1 comprises all functional segments. This study substantially improves the understanding of the generation of immunoglobulin diversity in cattle.

Improving mammalian genome scaffolding using large insert mate-pair next-generation sequencing

BackgroundPaired-tag sequencing approaches are commonly used for the analysis of genome structure. However, mammalian genomes have a complex organization with a variety of repetitive elements that complicate comprehensive genome-wide analyses.ResultsHere, we systematically assessed the utility of paired-end and mate-pair (MP) next-generation sequencing libraries with insert sizes ranging from 170 bp to 25 kb, for genome coverage and for improving scaffolding of a mammalian genome (Rattus norvegicus). Despite a lower library complexity, large insert MP libraries (20 or 25 kb) provided very high physical genome coverage and were found to efficiently span repeat elements in the genome. Medium-sized (5, 8 or 15 kb) MP libraries were much more efficient for genome structure analysis than the more commonly used shorter insert paired-end and 3 kb MP libraries. Furthermore, the combination of medium- and large insert libraries resulted in a 3-fold increase in N50 in scaffolding processes. Finally, we show that our data can be used to evaluate and improve contig order and orientation in the current rat reference genome assembly.ConclusionsWe conclude that applying combinations of mate-pair libraries with insert sizes that match the distributions of repetitive elements improves contig scaffolding and can contribute to the finishing of draft genomes.

Sequences Associated with Centromere Competency in the Human Genome

Centromeres, the sites of spindle attachment during mitosis and meiosis, are located in specific positions in the human genome, normally coincident with diverse subsets of alpha satellite DNA. While there is strong evidence supporting the association of some subfamilies of alpha satellite with centromere function, the basis for establishing whether a given alpha satellite sequence is or is not designated a functional centromere is unknown, and attempts to understand the role of particular sequence features in establishing centromere identity have been limited by the near identity and repetitive nature of satellite sequences. Utilizing a broadly applicable experimental approach to test sequence competency for centromere specification, we have carried out a genomic and epigenetic functional analysis of endogenous human centromere sequences available in the current human genome assembly. The data support a model in which functionally competent sequences confer an opportunity for centromere specification, integrating genomic and epigenetic signals and promoting the concept of context-dependent centromere inheritance.

Genomic Characterization of DArT Markers Based on High-Density Linkage Analysis and Physical Mapping to the Eucalyptus Genome

Diversity Arrays Technology (DArT) provides a robust, high throughput, cost-effective method to query thousands of sequence polymorphisms in a single assay. Despite the extensive use of this genotyping platform for numerous plant species, little is known regarding the sequence attributes and genome-wide distribution of DArT markers. We investigated the genomic properties of the 7,680 DArT marker probes of a Eucalyptus array, by sequencing them, constructing a high density linkage map and carrying out detailed physical mapping analyses to the Eucalyptus grandis reference genome. A consensus linkage map with 2,274 DArT markers anchored to 210 microsatellites and a framework map, with improved support for ordering, displayed extensive collinearity with the genome sequence. Only 1.4 Mbp of the 75 Mbp of still unplaced scaffold sequence was captured by 45 linkage mapped but physically unaligned markers to the 11 main Eucalyptus pseudochromosomes, providing compelling evidence for the quality and completeness of the current Eucalyptus genome assembly. A highly significant correspondence was found between the locations of DArT markers and predicted gene models, while most of the 89 DArT probes unaligned to the genome correspond to sequences likely absent in E. grandis, consistent with the pan-genomic feature of this multi-Eucalyptus species DArT array. These comprehensive linkage-to-physical mapping analyses provide novel data regarding the genomic attributes of DArT markers in plant genomes in general and for Eucalyptus in particular. DArT markers preferentially target the gene space and display a largely homogeneous distribution across the genome, thereby providing superb coverage for mapping and genome-wide applications in breeding and diversity studies. Data reported on these ubiquitous properties of DArT markers will be particularly valuable to researchers working on less-studied crop species who already count on DArT genotyping arrays but for which no reference genome is yet available to allow such detailed characterization.

Resolving the Breakpoints of the 17q21.31 Microdeletion Syndrome with Next-Generation Sequencing

Recurrent deletions have been associated with numerous diseases and genomic disorders. Few, however, have been resolved at the molecular level because their breakpoints often occur in highly copy-number-polymorphic duplicated sequences. We present an approach that uses a combination of somatic cell hybrids, array comparative genomic hybridization, and the specificity of next-generation sequencing to determine breakpoints that occur within segmental duplications. Applying our technique to the 17q21.31 microdeletion syndrome, we used genome sequencing to determine copy-number-variant breakpoints in three deletion-bearing individuals with molecular resolution. For two cases, we observed breakpoints consistent with nonallelic homologous recombination involving only H2 chromosomal haplotypes, as expected. Molecular resolution revealed that the breakpoints occurred at different locations within a 145 kbp segment of>99% identity and disrupt KANSL1 (previously known as KANSL1). In the remaining case, we found that unequal crossover occurred interchromosomally between the H1 and H2 haplotypes and that this event was mediated by a homologous sequence that was once again missing from the human reference. Interestingly, the breakpoints mapped preferentially to gaps in the current reference genome assembly, which we resolved in this study. Our method provides a strategy for the identification of breakpoints within complex regions of the genome harboring high-identity and copy-number-polymorphic segmental duplication. The approach should become particularly useful as high-quality alternate reference sequences become available and genome sequencing of individuals' DNA becomes more routine.

From expression cloning to gene modeling: The development of Xenopus gene sequence resources

The Xenopus community has made concerted efforts over the last 10–12 years systematically to improve the available sequence information for this amphibian model organism ideally suited to the study of early development in vertebrates. Here I review progress in the collection of both sequence data and physical clone reagents for protein coding genes. I conclude that we have cDNA sequences for around 50% and full-length clones for about 35% of the genes in Xenopus tropicalis, and similar numbers but a smaller proportion for Xenopus laevis. In addition, I demonstrate that the gaps in the current genome assembly create problems for the computational elucidation of gene sequences, and suggest some ways to ameliorate the effects of this. genesis 50:143–154, 2012. © 2012 Wiley Periodicals, Inc.

Upgraded genomic information of <i>Jatropha curcas</i> L.

In order to upgrade the genome sequence information of J. curcas L., we integrated de novo assembly of a total of 537 million paired-end reads generated from the Illumina sequencing platform into the current genome assembly which was obtained by a combination of the conventional Sanger method and the Roche/454 sequencing platform. The total length of the upgraded genome sequences thus obtained was 297,661,187 bp consisting of 39,277 contigs. The average and N50 lengths of the generated contigs were 7,579 bp and 15,950 bp, both of which were increased fourfold from the previous genome assembly. Along with genome sequence upgrading, the currently available transcriptome data were collected from the public databases and assembled into 19,454 tentative consensus sequences. Based on a comparison between these tentative consensus sequences of transcripts and the predictions of computer programs, a total of 30,203 complete and partial structures of protein-encoding genes were deduced. The number of genes with complete structures was increased about threefold from the previous genome annotation. By applying the upgraded genome sequence and predicted protein-coding gene information, the number and features of the tandemly arrayed genes, syntenic relations between Jatropha and other plant genomes, and structural features of transposable elements were investigated. The detailed information on the updated J. curcas genome is available at http://www.kazusa.or.jp/jatropha/.