Development of the endoderm and gut in medaka, Oryzias latipes

We performed an extensive analysis of endodermal development and gut tube morphogenesis in the medaka embryo by histology and in situ hybridization. The markers used in these analyses included sox17, sox32, foxA2, gata-4, -5, -6 and shh. sox17, sox32, foxA2, and gata-5 and -6 are expressed in the early endoderm to the onset of gut tube formation. Sections of medaka embryos hybridized with foxA2, a pan-endodermal marker during gut morphogenesis, demonstrated that gut tube formation is initiated in the anterior portion and that the anterior and mid/posterior gut undergo distinct morphogenetic processes. Tube formation in the anterior endoderm that is fated to the pharynx and esophagus is much delayed and appears to be independent of gut morphogenesis. The overall aspects of medaka gut development are similar to those of zebrafish, except that zebrafish tube formation initiates at both the anterior and posterior portions. Our results therefore describe both molecular and morphological aspects of medaka digestive system development that will be necessary for the characterization of medaka mutants.     

Complete mitochondrial genome of the marine medaka Oryzias melastigma (Beloniformes, Adrianichthyidae)

The complete mitochondrial genome was obtained from the assembled genome data sequenced by next-generation sequencer from the marine medaka Oryzias melastigma. The mitochondrial genome sequence was 16,864 bp in size, and the gene order and contents were identical with those of previously reported fish mitochondrial genomes. Of 13 protein-coding genes (PCGs), 4 genes (CO3, ND3, ND4, and Cytb) had incomplete stop codons. The base composition of O. melastigma mitogenome showed high A+T (59.65%) and anti-G bias (8.73%) on the 3rd position of PCGs.     

Development of the pancreas in medaka,Oryzias latipes,from embryo to adult

To address conserved and unique features of fish pancreas development, we performed extensive analyses of pancreatic development in medaka embryos and adults using pdx1‐ and ptf1a‐transgenic medaka, in situ hybridization and immunohistochemistry. The markers used in these analyses included pdx1, nkx6.1, nkx6.2, nkx2.2, Islet1, insulin, Somatostatin, glucagon, ptf1a, ela3l, trypsin, and amylase. The double transgenic (Tg) fish produced in the present study visualizes the development of endocrine (pdx1+) and exocrine (ptf1a+) parts simultaneously in living fishes. Like other vertebrates, the medaka pancreas develops as two (dorsal and ventral) buds in the anterior gut tube, which soon fuse into a single anlagen. The double Tg fish demonstrates that the differential property between the two buds is already established at the initial phase of bud development as indicated by strong pdx1 expression in the dorsal one. This Tg fish also allowed us to examine the gross morphology and the structure of adult pancreas and revealed unique characters of medaka pancreas such as broad and multiple connections with the gut tube along the anterior–posterior axis.     

Effects of pentachlorophenol on the reproduction of Japanese medaka (Oryzias latipes)

Pentachlorophenol (PCP) is widely used to control termites and protect wood from fungal-rot and wood-boring insects, and is often detected in the aquatic environment. Few studies have evaluated PCP as an environmental endocrine disruptor. In the present work, Japanese medaka (Oryzias latipes) was exposed to PCP for 28 days (F0 generation) with subsequent measurements of vitellogenin (VTG), hepatic 7-ethoxyresorufin-O-deethylase (EROD), and reproductive endpoints. Plasma VTG significantly increased in male fish treated with PCP concentrations lower than 200 microg/l and decreased in male and female animals exposed to 200 microg/l. Hepatic EROD from female fish increased when PCP exposure concentrations exceeded 20 microg/l, but decreased in the 200 microg/l PCP treatment group. Fecundity and mean fertility of female medaka decreased significantly in the second and third week following exposure concentrations greater than 100 microg/l, and testis-ova of male medaka was observed at PCP concentrations greater than 50 microg/l. Histological lesions of liver and kidney occurred when exposure concentrations exceeded 50 microg/l. In F1 generations, the hatching rates and time to hatch of offspring were significantly affected in fish exposed to 200 microg/l. These results indicated that PCP exposure caused responses consistent with estrogen and aryl hydrocarbon receptor activation as well as reproductive impairment at environmentally relevant concentrations.     

Analysis of haploid development based on expression patterns of developmental genes in the medaka Oryzias latipes

The abnormalities of haploid medaka embryos were characterized by comparative analysis of histologic sections and expression patterns of some developmental marker genes between haploids and diploids to clarify whether medaka haploids are useful for identifying mutants. During gastrulation, an obvious defect was first observed as a delay of epiboly and involution. This delay was shown to be caused not by the perturbation of mesoderm induction, but by widespread cell death and disorganization of cell arrangement in the blastoderm. This disorganization of cell arrangement was also detected in various organs, such as the brain, somite and notochord, at a late developmental stage. Ten days after fertilization, a small head and a short body axis were formed; these changes were also observed in haploid embryos in other species, but their cause is unknown. Based on the expression patterns of HNF3beta and goosecoid, it was demonstrated that a short and impotent prechordal plate induced near the marginal zone in haploid embryos was responsible for this defect. However, in these experiments it was also demonstrated that many major organs in haploids, such as the somite and notochord, differentiated incompletely but were present. Therefore, it was concluded that haploid screening is suitable for identifying mutations revealed by an obvious phenotype, such as dorsoventral polarity.     

The genome sequence of Samia ricini,a new model species of lepidopteran insect

Samia ricini, a gigantic saturniid moth, has the potential to be a novel lepidopteran model species. Samia ricini is far more resistant to diseases than the current model species Bombyx mori, and therefore can be more easily reared. In addition, genetic resources available for S. ricini rival those for B. mori: at least 26 ecoraces of S. ricini are reported and S. ricini can hybridize with wild Samia species, which are distributed throughout Asian countries, and produce fertile progenies. Physiological traits such as food preference, integument colour and larval spot pattern differ among S. ricini strains and wild Samia species so that those traits can be targeted in forward genetic analyses. To facilitate genetic research in S. ricini, we determined its whole genome sequence. The assembled genome of S. ricini was 458 Mb with 155 scaffolds, and the scaffold N50 length of the assembly was ~ 21 Mb. In total, 16,702 protein coding genes were predicted. While the S. ricini genome was mostly collinear with that of B. mori with some rearrangements and few S. ricini‐specific genes were discovered, chorion genes and fibroin genes seemed to have expanded in the S. ricini lineage. As the first step of genetic analyses, causal genes for “Blue,” “Yellow,” “Spot,” and “Red cocoon” phenotypes were mapped to chromosomes.     

Effect of 17-alpha-ethynylestradiol on germ cell proliferation in organ and primary culture of medaka (Oryzias latipes) testis

Organ cultures and primary cell cultures of medaka (Oryzias latipes) testis were compared with respect to cell viability and cell proliferation. The analysis by fluorescence microscopy and flow cytometry showed that in both cultures, the cells remained viable for at least 1 day and cell proliferation could be analyzed reliably by BrdU incorporation. The proliferating cells were mostly spermatogonia located at the periphery of the testis in tissue sections. Both culture systems were used to study the effect of 17-alpha-ethynylestradiol on cell proliferation. The results obtained with organ and primary cultures were consistent: low concentrations (0.01 and 1 nm) of synthetic estrogen stimulated cell proliferation slightly, while a higher concentration (100 nm) had an inhibitory effect. Both culture methods are suitable for the analysis of substances that might interfere with germ cell proliferation or other functions in spermatogenesis.     

The plastid genome of the red alga Laurencia

We present the 174,935 nt long plastid genome of the red alga Laurencia sp. JFC0032. It is the third plastid genome characterized for the largest order of red algae (Ceramiales). The circular‐mapping plastid genome is small compared to most florideophyte red algae, and our comparisons show a trend toward smaller plastid genome sizes in the family Rhodomelaceae, independent from a similar trend in Cyanidiophyceae. The Laurencia genome is densely packed with 200 annotated protein‐coding genes (188 widely conserved, 3 open reading frames shared with other red algae and 9 hypothetical coding regions). It has 29 tRNAs, a single‐copy ribosomal RNA cistron, a tmRNA, and the RNase P RNA.     

Whole‐exome targeted sequencing of the uncharacterized pine genome

The large genome size of many species hinders the development and application of genomic tools to study them. For instance, loblolly pine (Pinus taeda L.), an ecologically and economically important conifer, has a large and yet uncharacterized genome of 21.7 Gbp. To characterize the pine genome, we performed exome capture and sequencing of 14 729 genes derived from an assembly of expressed sequence tags. Efficiency of sequence capture was evaluated and shown to be similar across samples with increasing levels of complexity, including haploid cDNA, haploid genomic DNA and diploid genomic DNA. However, this efficiency was severely reduced for probes that overlapped multiple exons, presumably because intron sequences hindered probe:exon hybridizations. Such regions could not be entirely avoided during probe design, because of the lack of a reference sequence. To improve the throughput and reduce the cost of sequence capture, a method to multiplex the analysis of up to eight samples was developed. Sequence data showed that multiplexed capture was reproducible among 24 haploid samples, and can be applied for high‐throughput analysis of targeted genes in large populations. Captured sequences were de novo assembled, resulting in 11 396 expanded and annotated gene models, significantly improving the knowledge about the pine gene space. Interspecific capture was also evaluated with over 98% of all probes designed from P. taeda that were efficient in sequence capture, were also suitable for analysis of the related species Pinus elliottii Engelm.     

The ultrastructure of the ovarian follicle of medaka,Oryzias latipes

Summary The follicular cells in the oocytes of Oryzias latipes were studied by electron microscopy in order to clarify the fine structure, and the role of the cells during yolk formation and ovulation. The smallest follicles were observed during the early phase of peri-nucleolus stage of the oocyte. The cells have flattened nuclei, and perikarya with undeveloped organelles. But when the oocytes attain diameter of about 250 (yolk vesicle stage), both types of endoplasmic reticula are present. Moreover, the microvilli of the plasma membrane of oocyte as well as the follicles protrude into the pore canals of the zona radiata. In the oocytes of yolk stage the rough-surfaced endoplasmic-reticulum is typically developed and observed around the nuclei. Other organelles (lysosomes, mitochondria and Golgi) increase in number. The relation between the changes of cytoarchitecture in the follicles and yolk formation is discussed.At 17.00 p.m. on the day preceding ovulation the microvilli withdraw somewhat. Ribosomes are attached to the vesicular and cisternal endoplasmic reticula. When the oocytes attain complete maturation (24.00 p.m. at near ovulation), striking changes of the follicles are observed. The microvilli are almost withdrawn. In the degenerating follicles the lamellar structure is formed, and lipids are deposited at the center. At this time the contents of lysosomes have mostly disappeared.     

Construction and comparison of three reference‐quality genome assemblies for soybean

We report reference‐quality genome assemblies and annotations for two accessions of soybean (Glycine max) and for one accession of Glycine soja, the closest wild relative of G. max. The G. max assemblies provided are for widely used US cultivars: the northern line Williams 82 (Wm82) and the southern line Lee. The Wm82 assembly improves the prior published assembly, and the Lee and G. soja assemblies are new for these accessions. Comparisons among the three accessions show generally high structural conservation, but nucleotide difference of 1.7 single‐nucleotide polymorphisms (snps) per kb between Wm82 and Lee, and 4.7 snps per kb between these lines and G. soja. snp distributions and comparisons with genotypes of the Lee and Wm82 parents highlight patterns of introgression and haplotype structure. Comparisons against the US germplasm collection show placement of the sequenced accessions relative to global soybean diversity. Analysis of a pan‐gene collection shows generally high conservation, with variation occurring primarily in genomically clustered gene families. We found approximately 40–42 inversions per chromosome between either Lee or Wm82v4 and G. soja, and approximately 32 inversions per chromosome between Wm82 and Lee. We also investigated five domestication loci. For each locus, we found two different alleles with functional differences between G. soja and the two domesticated accessions. The genome assemblies for multiple cultivated accessions and for the closest wild ancestor of soybean provides a valuable set of resources for identifying causal variants that underlie traits for the domestication and improvement of soybean, serving as a basis for future research and crop improvement efforts for this important crop species.     

Fish cell culture: Establishment of two fibroblast-like cell lines (OL-17 and OL-32) from fins of the medaka,Oryzias latipes

Summary Two fibroblast-like cell lines were obtained from fins of adults of the medaka,Oryzias latipes, and serially cultured at 27° C. One cell line, which was derived from a fish of the orange-red variety, reached to population doubling level (PDL) 434 on Day 840 by 120 passages. The other, which was derived from a fish of the inbred strain HB32C, reached to PDL 294 on Day 551 by 80 passages. Any symptom of crisis was not detected. The cell lines were named OL-17 and OL-32, respectively. Population doubling time was 29 h for OL-17 cells, and 32 h for OL-32 cells. Density-dependent inhibition of growth was clear in OL-32 cells, but not so obvious in OL-17 cells. Modal chromosome number of OL-17 cells was 50, and that of OL-32 cells was 47 (2N=48). Plating efficiency of OL-17 cells was about 10%, whereas that of OL-32 cells was about 5%. The use of conditioned medium (80% concentration) increased the plating efficiency of OL-32 cells to more than 25%.     

Improved genome assembly provides new insights into genome evolution in a desert poplar (Populus euphratica)

Populus euphratica is well adapted to extreme desert environments and is an important model species for elucidating the mechanisms of abiotic stress resistance in trees. The current assembly of P. euphratica genome is highly fragmented with many gaps and errors, thereby impeding downstream applications. Here, we report an improved chromosome‐level reference genome of P. euphratica (v2.0) using single‐molecule sequencing and chromosome conformation capture (Hi‐C) technologies. Relative to the previous reference genome, our assembly represents a nearly 60‐fold improvement in contiguity, with a scaffold N50 size of 28.59 Mb. Using this genome, we have found that extensive expansion of Gypsy elements in P. euphratica led to its rapid increase in genome size compared to any other Salicaceae species studied to date, and potentially contributed to adaptive divergence driven by insertions near genes involved in stress tolerance. We also detected a wide range of unique structural rearrangements in P. euphratica, including 2,549 translocations, 454 inversions, 121 tandem and 14 segmental duplications. Several key genes likely to be involved in tolerance to abiotic stress were identified within these regions. This high‐quality genome represents a valuable resource for poplar breeding and genetic improvement in the future, as well as comparative genomic analysis with other Salicaceae species.