Matthew D. Clark

A chromosome conformation capture ordered sequence of the barley genome

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

Zebrafish Dkk1, induced by the pre-MBT Wnt signaling, is secreted from the prechordal plate and patterns the anterior neural plate

mRNA injection into the ventral blastomeres of Xenopus embryos of mRNA encoding Wnt pathway genes induces a secondary axis with complete head structures. To identify target genes of the pre-MBT dorsalization pathway that might be responsible for head formation in zebrafish, we have cloned zebrafish dickkopf1 (dkk1), which is expressed in tissues implicated in head patterning. We found that dkk1 blocks the post-MBT Wnt signaling and dkk1 is a target of the pre-MBT Wnt signaling. Dkk1 overexpression in the prechordal plate suggests that Dkk1, secreted from the prechordal plate, expands the forebrain at the expense of the midbrain in the anterior neural plate. Furthermore, dkk1 acts in parallel to the homeobox gene bozozok and bozozok is required for the maintenance of dkk1 expression. The nodal gene squint is also required for the maintenance of dkk1 expression. Among the mutually dependent target genes of the pre-MBT Wnt signaling, dkk1 plays an important role in patterning the anterior head of zebrafish.

NextClip: an analysis and read preparation tool for Nextera long mate pair libraries

SUMMARY Illuminas recently released Nextera Long Mate Pair (LMP) kit enables production of jumping libraries of up to 12 kb. The LMP libraries are an invaluable resource for carrying out complex assemblies and other downstream bioinformatics analyses such as the characterization of structural variants. However, LMP libraries are intrinsically noisy and to maximize their value, post-sequencing data analysis is required. Standardizing laboratory protocols and the selection of sequenced reads for downstream analysis are non-trivial tasks. NextClip is a tool for analyzing reads from LMP libraries, generating a comprehensive quality report and extracting good quality trimmed and deduplicated reads. AVAILABILITY AND IMPLEMENTATION Source code, user guide and example data are available from https://github.com/richardmleggett/nextclip/.

An improved assembly and annotation of the allohexaploid wheat genome identifies complete families of agronomic genes and provides genomic evidence for chromosomal translocations

Advances in genome sequencing and assembly technologies are generating many high-quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimized data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents >78% of the genome with a scaffold N50 of 88.8 kb that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNA-seq and Pacific Biosciences (PacBio) full-length cDNAs to identify 104,091 high-confidence protein-coding genes and 10,156 noncoding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop.

AmphiBMP2/4, an amphioxus bone morphogenetic protein closely related to Drosophila decapentaplegic and vertebrate BMP2 and BMP4: Insights into evolution of dorsoventral axis specification

Amphioxus AmphiBMP2/4 appears to be a single gene closely related to vertebrate BMP2and BMP4. In amphioxus embryos, the expression patterns of AmphiBMP2/4 suggest patterning roles in the ectodermal dorsoventral axis (comparable to dorsoventral axis establishment in the ectoderm by Drosophila decapentaplegic and vertebrate BMP4). In addition AmphiBMP2/4 may be involved in somite evagination, tail bud growth, pharyngeal differentiation (resulting in club‐shaped gland morphogenesis), hindgut regionalization, differentiation of olfactory epithelium, patterning of the anterior central nervous system, and establishment of the heart primordium. One difference between the developmental role of amphioxus AmphiBMP2/4 and vertebrate BMP4is that the former does not appear to be involved in the initial establishment of the dorsoventral polarity of the mesoderm. Dev. Dyn. 1998;213:130–139.

Genetic screens for mutations affecting development of Xenopus tropicalis.

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics.

Construction and analysis of arrayed cDNA libraries.

For any attempt to understand the biology of an organism the incorporation of a cDNA-based approach is unavoidable, because it is a major approach to studying gene function. The complete sequence of the genome alone is not sufficient to understand any organism; its gene regulation, expression, splice variation, posttranslational modifications, and protein-protein interactions all need to be addressed. Because the majority of vertebrate genes have probably been identified as ESTs the next stage of the Human Genome Project is attributing functional information to these sequences. In most cases hybridization-based approaches on arrayed pieces of DNA represent the most efficient way to study the expression level and splicing of a gene in a given tissue. Similar technology, now being applied at the protein level using protein expression libraries, high-density protein membranes, and antibody screening, should allow studies of protein localization and modifications. Coupled to these approaches is the use of technologies, which although lacking the highly parallel nature of hybridization, can potentially characterize large numbers of samples individually and with high accuracy. Automated gel-based DNA sequencing is an example of such a technique; protein sequencing and mass fingerprinting are further examples. In the case of mass spectroscopic analysis, the speed and sensitivity are vastly superior to that of gel-based approaches; however, the preparation of samples is more tedious. Our laboratory is developing a system to characterize DNA samples by mass spectrometry, allowing more rapid genotyping than is currently possible using gel-based technologies ([symbol: see text]. Gut, [symbol: see text]. Berlin and H. Lehrach, personal communication, 1998). Such technology would make information on gene polymorphisms widely accessible. Data generated using all of these techniques at the DNA and protein level will be connected by both protein expression libraries and database comparisons; finally, two hybrid library screens will identify many of the protein-protein interactions, linking genes together. In this way we will start to understand the interplay between genes on a global scale, both at the level of molecular interaction and the biological processes they regulate.

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO2. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.

Accelerated cloning of a potato late blight-resistance gene using RenSeq and SMRT sequencing

Global yields of potato and tomato crops have fallen owing to potato late blight disease, which is caused by Phytophthora infestans. Although most commercial potato varieties are susceptible to blight, many wild potato relatives show variation for resistance and are therefore a potential source of Resistance to P. infestans (Rpi) genes. Resistance breeding has exploited Rpi genes from closely related tuber-bearing potato relatives, but is laborious and slow. Here we report that the wild, diploid non-tuber-bearing Solanum americanum harbors multiple Rpi genes. We combine resistance (R) gene sequence capture (RenSeq) with single-molecule real-time (SMRT) sequencing (SMRT RenSeq) to clone Rpi-amr3i. This technology should enable de novo assembly of complete nucleotide-binding, leucine-rich repeat receptor (NLR) genes, their regulatory elements and complex multi-NLR loci from uncharacterized germplasm. SMRT RenSeq can be applied to rapidly clone multiple R genes for engineering pathogen-resistant crops.

Genomic innovation for crop improvement

Crop production needs to increase to secure future food supplies, while reducing its impact on ecosystems. Detailed characterization of plant genomes and genetic diversity is crucial for meeting these challenges. Advances in genome sequencing and assembly are being used to access the large and complex genomes of crops and their wild relatives. These have helped to identify a wide spectrum of genetic variation and permitted the association of genetic diversity with diverse agronomic phenotypes. In combination with improved and automated phenotyping assays and functional genomic studies, genomics is providing new foundations for crop-breeding systems.