Cytological and genetical studies on male sterility inCryptomeria japonica D. Don

Genetic male sterility is a useful trait in plant breeding, especially in angiosperm crops such as corn, onion and carrot. We found a male sterile sugi (Cryptomeria japonica D. Don) tree in Toyama, Japan. Pollen of sugi is one of the major causes of pollinosis in Japan. We carried out this research in an attempt to make clear the characteristics and inheritance of this male sterility. Microsporogenesis of the male sterile tree proceeded meiosis, however, the microspores collapsed after they were separated from pollen tetrads in locules, resulting in complete male sterility. Most likely, ethylene evolution was responsible for male sterility expression. Full seed setting in the male sterile tree indicated normal macrosporogenesis. Seeds obtained from crossing between male sterile and normal lines showed relatively high level of germination and their seedlings grew vigorously. The somatic chromosome numbers of 241 germinated seeds, derived from the male sterile tree, were mostly 22, euploid. These results indicated that male sterile tree was different from other similar previously reported trees with low pollen fertility, resulting from triploid or trisomics. Probably, male sterility in sugi is either nuclear genetic male sterility or cytoplasmic male sterility. The study was partially supported by Program for Promotion of Basic Research Actives for Innovative Biosciences.     

The functional effect of Gly209 and Ile213 substitutions on lysozyme activity of family 19 chitinase encoded by cyanophage Ma-LMM01

ORF69 in the cyanophage infecting Microcystis aeruginosa, Ma-LMM01, shows homology to the family 19 chitinases where the catalytic domain has structural similarity to lysozyme. Chitinases hydrolyze chitin, a β-1, 4-linked monopolymer of N-acetylglucosamine (GlcNAc); whereas lysozymes hydrolyzes peptidoglycan, alternating β-1, 4-linked copolymers of N-acetylmuramic acid (MurNAc) and GlcNAc. Using amino acid sequence comparison to ORF69, the putative sugar binding residues, Gln162 and Lys165, from the barley chitinase (the model enzyme for the family 19 chitinases) corresponding to subsites −4 and −3 were found to be replaced with Gly209 and Ile213, respectively, in ORF69. To analyze their contribution to substrate binding affinity, ORF69 was cloned into Escherichia coli; and two mutant proteins G209Q and I213K were prepared using site-directed mutagenesis. The wild-type gene product (gp69) showed both lysozyme and chitinase activities. In contrast, the I213K mutant showed a decrease (70%) in lysozyme activity and a significant increase (50%) in chitinase activity; whereas, the G209Q mutant almost completely abolished both enzyme activities. The data suggest the Ile213 residue is involved in recognizing the substrate MurNAc; and Gly209 has significant contribution in chitinase and lysozyme activities for the wild-type gp69.     

Successive changes of hematologic characteristics and plasma chemistry values of juvenile loggerhead turtles (Caretta caretta).

Hematologic characteristics and plasma chemistry values of juvenile loggerhead turtles (Caretta caretta) from the ages of 1 mo to 3 yr were obtained to establish baseline values. Five clinically normal loggerhead turtles were selected from the same clutch and raised in an indoor artificial nesting beach. Blood samples were successively collected and examined for various blood characteristics for a maximum total of 15 times. Hematologic characteristics, including packed cell volume, white blood cell counts, and white blood cell differentials; and plasma chemistry values, including total bilirubin, total protein, albumin, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, gamma-glutamic transpeptidase, creatinine, blood urea nitrogen, uric acid, alkaline phosphatase, amylase, triglyceride, total cholesterol, ionized sodium, ionized potassium and ionized chlorine, were measured. These results were used to establish a hematology and blood chemistry baseline for captive juvenile loggerhead turtles and will aid in their medical management.     

Sequence and polymerase chain reaction–restriction fragment length polymorphism analysis of the large subunit rRNA gene of bivalve: Simple and widely applicable technique for multiple species identification of bivalve larva

ABSTRACT:   One of the biggest and long-standing difficulties in investigation of larval ecology in the field is species-level identification. In the present study, we developed polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis based on the large subunit (LSU) rRNA gene (rDNA) D1/D2/D3 region for identification of multiple species of bivalve larvae using 14 species of bivalve collected from Maizuru Bay. The LSU rDNA D1/D2/D3 region of all analyzed species could be amplified by PCR using bvD1f/bvD3r primers, and RFLP analysis by Hae III digestion on the PCR products showed species-specific fragment patterns. Furthermore, this analysis applied to single bivalve larvae in Maizuru Bay revealed efficient amplification of the target region and the species-specific pattern from 80% of the larvae, 75% of which showed a pattern that matched a certain pattern of the adult bivalves. In addition, the analysis of inter- and intraspecies variation of the LSU rDNA D1/D2/D3 region using the sequence data of the genus Crassostrea from the DDBJ database showed high applicability of this RFLP analysis on closely related species. Because of the wide applicability and technical simplicity, this method can become the standard for the identification of bivalve larvae species once the sequence data of the LSU rDNA D1/D2/D3 region of many bivalve species have been accumulated.     

Activation with ethanol improves embryo development of ICSI-derived oocytes by regulation of kinetics of MPF activity

Developmental potential of bovine embryos that are not artificially activated after intracytoplasmic sperm injection (ICSI) is generally very low. In this study, we investigated effects of artificial activation with ethanol on kinetics of maturation promoting factor (MPF) activity (p34(cdc2) kinase activity) and development of bovine oocytes following ICSI. Treatment of oocytes with ethanol at 4 h after ICSI improved their first cleavage and further preimplantation development (51% vs. 13%, 14% vs. 4%: treatment with vs. without ethanol, respectively). MPF activity of oocytes was lowered until at least 2 h after ICSI. In oocytes without activation after ICSI, MPF activity temporarily elevated at 6 h after ICSI, whereas this phenomena was not observed in the oocytes treated with ethanol. Furthermore, MPF activity was elevated 20 h after ICSI in oocytes activated with ethanol, whereas this elevation of MPF activity was not shown in oocytes without activation. These results indicate that the stimulus of sperm was sufficient to lower MPF activity of oocytes following ICSI, and moreover the activation treatment of bovine oocytes with ethanol after ICSI served to maintain the low levels of MPF activity until the next cell cycle started.     

Verification of a phenotypic discrimination method for hybrid larch seedlings using DNA markers

The hybrid between Kuril larch (Larix gmelinii var. japonica) and Japanese larch (L. kaempferi) is an important afforestation tree species in Hokkaido, Japan, because of its rapid juvenile growth, straight stem, and resistance to bark gnawing by voles. To produce desirable hybrid seedlings, precise seedling discrimination is essential. However, continuous variations in morphological and phenological traits occur across L. gmelinii var. japonica × L. kaempferi and L. gmelinii var. japonica × L. gmelinii var. japonica seedlings. Therefore, we used DNA markers to verify and improve the morphological and phenological discrimination method. We collected seeds from an interspecific seed orchard and, using chloroplast DNA analysis, we showed that the hybridization rates of 1-year-old seedlings were different between years (2004, 23.2 %; 2005, 53.6 %) and between mother trees (2004, 5.8–39.4 %; 2005, 20.0–81.0 %). Discriminant analyses revealed that the root collar diameter of 2-year-old seedlings, number of sylleptic branches of 2-year-old seedlings, and day of terminal bud set in 1- and 2-year-old seedlings are traits that aid in discriminating hybrid seedlings. The proportions of correctly discriminated individuals were found to be 81.7–88.2 % when using improved phenotypic discrimination methods. These methods adopted traits with high discrimination abilities during discriminant analyses and determined the selection intensity according to the hybridization rate estimated from the DNA markers. In contrast, the proportions of correctly discriminated individuals obtained using the current method were lower, 72.7–78.5 %, because this method was based on seedling height and adopted a constant selection intensity.     

Genetic evidence for paternal inheritance of the chloroplast in four Australian Callitris species (Cupressaceae)

The mode of chloroplast inheritance was investigated in the four Australian conifer species; Callitris columellaris, C. intratropica, C. gracilis and C. verrucosa (Callitroideae, Cupressaceae), as a basis for future use of chloroplast genetic markers for the genus. Open-pollinated seeds and the foliage tissue of the mother trees were collected from one population per species, in which two distinct chloroplast haplotypes at the trnL (UAA) 5′ exon–trnF (GAA) region were co-distributed. In total, the DNA sequences of the chloroplast fragments in 255 samples were determined, and the haplotypes of the germinated seedlings and the mother trees were compared. Among the 16 mother trees examined across all species, 12 trees were shown to have produced at least one offspring with a different haplotype from the mother, indicating the occurrence of paternal inheritance of chloroplast in the fertilization processes. In binomial GLMM analyses, the haplotype of mother trees in C. columellaris, C. gracilis, and C. verrucosa did not significantly affect those of their offspring, and the predicted frequency of seedlings’ haplotypes was compatible with the observed frequency in adult canopy trees. These findings provide a genetic support that paternal inheritance of chloroplast DNA via pollen dispersal would be dominant in the Callitris species.     

Induction of (<Emphasis Type="Italic">Z</Emphasis>)-8-heptadecene in marine green alga <Emphasis Type="Italic">Bryopsis maxima</Emphasis> by mechanical wounding

ABSTRACT:   After mechanical wounding of the marine green alga Bryopsis maxima in a phosphate buffer, one main organic component was released to the solution. The organic compound was characterized by a molecular ion at 238  m/z by gas chromatography–mass spectrometry (GC-MS) and was deduced as 8-heptadecene by the fragment pattern. To identify the stereochemistry of 8-heptadecene, synthetic ( Z / E )-8-heptadecene was coinjected to the solution of wounded B. maxima . Comparing retention times and mass spectra with the synthetic sample, the target hydrocarbon obtained from wounded B. maxima was coincident with ( Z )-8-heptadecene.     

Phylogenetic analysis of noxious red tide flagellates Chattonella antiqua, C. marina, C. ovata, and C. verruculosa (Raphidophyceae) based on the rRNA gene family

ABSTRACT:   Four species of Chattonella , which are well known to form red tides that are lethal to fish, were subjected to phylogenetic analysis on the basis of the ribosomal RNA genes (rDNA), 5.8S rDNA, 18S rDNA, 28S rDNA, and the flanking internal transcribed spacers 1 and 2 (ITS1 and ITS2). The 18S rDNA sequences of C. antiqua , C. marina , and C. ovata isolated from different regions in Japan were compared. They were found to be identical with each other in a sequence 1818 bp long. The sequences of the D1/D2 region in the 28S rDNA, 5.8S rDNA, and ITS region that are known to be more variable regions were also found to be identical. These homogeneities of the rRNA gene family revealed the extremely close relatedness of C. antiqua , C. marina , and C. ovata . The sequences of C. verruculosa were different from those of these three species , resulting in an 89.2% homology in the 18S rDNA sequences, 70.4% homology in the D1/D2 region in the 28S rDNA sequences, and an 81.5% homology in 5.8S rDNA sequences and the ITS regions. Chattonella verruculosa was grouped within a single cluster composed of Dictyochophyceae rather than the other species of Raphidophyceae.     

Improvement of application method of liquid fertilizer with irrigation water for uniform topdressing and change in nitrogen concentration of flooded water after application

(pp. 811–816)
The application method of liquid fertilizer with irrigation water was tested in large scale paddy fields using a newly developed fertilizer supplier which can make a supplying rate of liquid fertilizer constant. Uniformity of fertilization, change in the nitrogen concentration of flooded water and absorption of applied nitrogen by paddy rice were examined. High uniformity of fertilization was achieved with this method as compared with a conventional broadcast application of granular fertilizer, or an inflow fertilization of granule-like fertilizer with irrigation water.
When urea was applied as liquid fertilizer, the fertilizer-N disappeared quickly from ponding water under shallow conditions of ponding water depth. In the case of the same depth of ponding water, the rate of reduction was large under conditions of large permeability of water.
The nitrogen utilization rates of urea and ammonium sulfate labeled with ¹⁵N were not related to the depth of ponding water at the time of fertilization. When ammonium sulfate was supplied the nitrogen utilization rate was around 50% regardless of fertilization conditions however, the urea-N utilization rate fell to 40% or less at a permeability of ponding water of 0.8 cm day⁻¹ or less, while it increased to 50% equivalent to ammonium sulfate, at 0.8 cm day⁻¹ or more permeability.