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High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.04.006 Byline: Donald A. Yergeau (a), Catharine N. Cornell (a), Sandra K. Parker (a), Yi Zhou (b), H. William Detrich (a) Abstract: The Antarctic icefishes (family Channichthyidae, suborder Notothenioidei) constitute the only vertebrate taxon that fails to produce red blood cells. These fishes can be paired with closely related, but erythrocyte-producing, notothenioids to discover erythropoietic genes via representational difference analysis. Using a B30.2-domain-encoding DNA probe so derived from the hematopoietic kidney (pronephros) of a red-blooded Antarctic rockcod, Notothenia coriiceps, we discovered a related, novel gene, bloodthirsty (bty), that encoded a 547-residue protein that contains sequential RING finger, B Box, coiled-coil, and B30.2 domains. bty mRNA was expressed by the pronephric kidney of N. coriiceps at a steady-state level 10-fold greater than that found in the kidney of the icefish Chaenocephalus aceratus. To test the function of bty, we cloned the orthologous zebrafish gene from a kidney cDNA library. Whole-mount in situ hybridization of zebrafish embryos showed that bty mRNA was present throughout development and, after the mid-blastula transition, was expressed in the head and in or near the site of primitive erythropoiesis in the tail just prior to red cell production. One- to four-cell embryos injected with two distinct antisense morpholino oligonucleotides (MOs) targeted to the 5'-end of the bty mRNA failed to develop red cells, whereas embryos injected with 4- and 5-bp mismatch control MOs produced wild-type quantities of erythrocytes. The morphant phenotype was rescued by co-injection of synthetic bty mRNA containing an artificial 5'-untranslated region (UTR) with the antisense MO that bound the 5'-UTR of the wild-type bty transcript. Furthermore, the expression of genes that mark terminal erythroid differentiation was greatly reduced in the antisense-MO-treated embryos. We conclude that bty is likely to play a role in differentiation of the committed red cell progenitor. Author Affiliation: (a) Department of Biology, 134 Mugar Hall, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA (b) Children's Hospital and Howard Hughes Medical Institute, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA

The potential contribution of posttranslational modifications, in particular glutamylation of the C-termini of the tubulin chains, to the cold-assembling phenotype of the microtubules of Antarctic fishes is investigated. It is suggested that the unique residue substitutions in the primary structures of Antarctic fish tubulin isotypes and quantitative changes in isoform glutamylation act synergistically to adapt microtubule assembly to low temperatures.

Ancestors of the Antarctic icefishes (family Channichthyidae) were benthic and had no swim bladder, making it energetically expensive to rise from the ocean floor. To exploit the water column, benthopelagic icefishes were hypothesized to have evolved a skeleton with "reduced bone," which gross anatomical data supported. Here, we tested the hypothesis that changes to icefish bones also occurred below the level of gross anatomy. Histology and micro‐CT imaging of representative craniofacial bones (i.e., ceratohyal, frontal, dentary, and articular) of extant Antarctic fish species specifically evaluated two features that might cause the appearance of "reduced bone": bone microstructure (e.g., bone volume fraction and structure linear density) and bone mineral density (BMD, or mass of mineral per volume of bone). Measures of bone microstructure were not consistently different in bones from the icefishes Chaenocephalus aceratus and Champsocephalus gunnari, compared to the related benthic notothenioids Notothenia coriiceps and Gobionotothen gibberifrons. Some quantitative measures, such as bone volume fraction and structure linear density, were significantly increased in some icefish bones compared to homologous bones of non‐icefish. However, such differences were rare, and no microstructural measures were consistently different in icefishes across all bones and species analyzed. Furthermore, BMD was similar among homologous bones of icefish and non‐icefish Antarctic notothenioids. In summary, "reduced bone" in icefishes was not due to systemic changes in bone microstructure or BMD, raising the prospect that "reduced bone" in icefish occurs only at the gross anatomic level (i.e., smaller or fewer bones). Given that icefishes exhibit delayed skeletal development compared to non‐icefish Antarctic fishes, combining these phenotypic data with genomic data might clarify genetic changes driving skeletal heterochrony. [ABSTRACT FROM AUTHOR]

In vertebrates, hematopoietic and vascular progenitors develop from ventral mesoderm. The first primitive wave of hematopoiesis yields embryonic red blood cells, whereas progenitor cells of subsequent definitive waves form all hematopoietic cell lineages. In this report we examine the development of hematopoietic and vasculogenic cells in normal zebrafish and characterize defects in cloche and spadetail mutant embryos. The zebrafish homologs of lmo2, c-myb, fli1, flk1, and flt4 have been cloned and characterized in this study. Expression of these genes identifies embryonic regions that contain hematopoietic and vascular progenitor cells. The expression of c-myb also identifies definitive hematopoietic cells in the ventral wall of the dorsal aorta. Analysis of b316 mutant embryos that carry a deletion of the c-myb gene demonstrates that c-myb is not required for primitive erythropoiesis in zebrafish even though it is expressed in these cells. Both cloche and spadetail mutant embryos have defects in primitive hematopoiesis and definitive hematopoiesis. The cloche mutants also have significant decreases in vascular gene expression, whereas spadetail mutants expressed normal levels of these genes. These studies demonstrate that the molecular mechanisms that regulate hematopoiesis and vasculogenesis have been conserved throughout vertebrate evolution and the clo and spt genes are key regulators of these programs. Key Words: zebrafish; hematopoiesis; blood; Danio rerio.

The structural and functional characteristics of inner and outer arm dyneins isolated from sperm flagella of the Antarctic rockcod Notothenia coriiceps were investigated. The inner arm dynein complex was found to be similar in subunit composition to the flagellar inner arm dyneins of Chlamydomonas. Also, a single-copy gene for an inner arm dynein heavy chain (DHC) was found. The outer arm complex has two DHCs and five intermediate chains.

For mammals [[Beta].sub.2]-microgiobulin ([[Beta].sub.2]m), the light chain of major histocompatibility complex (MHC) class I molecules, is invariant (or highly conserved) and is encoded by a single gene unlinked to the MHC. We find that [[Beta].sub.2]m of a saimonid fish, the rainbow trout (Oncorhynchus mykiss), does not conform to the mammalian paradigm. Ten of 12 randomly selected [[Beta].sub.2]m cDNA clones from an individual fish have different nucleotide sequences. A complex restriction fragment length polymorphism pattern is observed with rainbow trout, suggesting multiple [[Beta].sub.2]m genes in the genome, in excess of the two genes expected from the ancestral salmonid tetraploidy. Additional duplication and diversification of the [[Beta].sub.2]m genes might have occurred subsequently. Variation in the [[Beta].sub.2]m cDNA sequences is mainly at sites that do not perturb the structure of the mature [[Beta].sub.2]m protein, showing that the observed diversity of the trout [[Beta].sub.2]m genes is not primarily a result of pathogen selection.

A search of the genomes of three white-blooded icefishes and four red-blooded Notothenioidei relatives for the presence of globin-related DNA sequences reveals the hybridization of the alpha-globin probe to red-and white-blooded species genomic DNAs, while hybridzation of the beta-globin probe occurs with only red-blooded species DNAs. The inactive genomic remains of alpha-globin genes are maintained by the ice-fishes, while the beta-globin-encoding gene is lost through rapid mutation or deletion, accounting for the hemoglobinless phenotypes of extant icefishes.

Proteolysis or neutralization of carboxyl groups at the C-termini of Antarctic fish neutral tubulins enables analysis of the polymerization process. The microtubule with an altered C-termini due to neutralization assembles easily when compared to control tubulin. The critical concentration of the untreated protein is more than that of the modified one, indicating that the C-termini is not involved in polymerization.