A quantitative protocol for DNA metabarcoding of springtails (Collembola).

We developed a novel protocol with superior quantitative analysis results for DNA metabarcoding of Collembola, a major soil microarthropod order. Degenerate PCR primers were designed for conserved regions in the mitochondrial cytochrome c oxidase subunit I (mtCOI) and 16S ribosomal RNA (mt16S) genes based on published collembolan mitogenomes. The best primer pair was selected based on its ability to amplify each gene, irrespective of the species. DNA was extracted from 10 natural communities sampled in a temperate forest (with typically 25-30 collembolan species per 10 soil samples) and 10 mock communities (with seven cultured collembolan species). The two gene regions were then amplified using the selected primers, ligated with adapters for 454 technology, and sequenced. Examination of the natural community samples showed that 32 and 36 operational taxonomic units (defined at a 90% sequence similarity threshold) were recovered from the mtCOI and mt16S data, respectively, which were comparable to the results of the microscopic identification of 25 morphospecies. Further, sequence abundances for each collembolan species from the mtCOI and mt16S data of the mock communities, after normalization by using a species as the internal control, showed good correlation with the number of individuals in the samples (R = 0.91-0.99), although relative species abundances within a mock community sample estimated from sequences were skewed from community composition in terms of the number of individuals or biomass of the species. Thus, this protocol enables the comparison of collembolan communities in a quantitative manner by metabarcoding.

Gene expression analysis of disabled and re-induced isoprene emission by the tropical tree Ficus septica before and after cold ambient temperature exposure.

Isoprene is the most abundant type of nonmethane, biogenic volatile organic compound in the atmosphere, and it is produced mainly by terrestrial plants. The tropical tree species Ficus septica Burm. F. (Rosales: Moraceae) has been shown to cease isoprene emissions when exposed to temperatures of 12 °C or lower and to re-induce isoprene synthesis upon subsequent exposure to temperatures of 30 °C or higher for 24 h. To elucidate the regulation of genes underlying the disabling and then induction of isoprene emission during acclimatization to ambient temperature, we conducted gene expression analyses of F. septica plants under changing temperature using quantitative real-time polymerase chain reaction and western blotting. Transcription levels were analyzed for 17 genes that are involved in metabolic pathways potentially associated with isoprene biosynthesis, including isoprene synthase (ispS). The protein levels of ispS were also measured. Changes in transcription and protein levels of the ispS gene, but not in the other assessed genes, showed identical temporal patterns to isoprene emission capacity under the changing temperature regime. The ispS protein levels strongly and positively correlated with isoprene emission capacity (R(2) = 0.92). These results suggest that transcriptional regulation of ispS gave rise to the temporal variation in isoprene emission capacity in response to changing temperature.

Genomic sequencing-based detection of large deletions in Rhodococcus rhodochrous strain B-276.

Bacteria of the genus Rhodococcus (Actinomycetes) have the ability to catabolize various organic compounds and are therefore considered potential genetic resources for applications such as bioremediation. We investigated a next-generation sequencing-based procedure to rapidly identify candidate functional gene(s) from rhodococci on the basis of their frequent genome recombination. The Rhodococcus rhodochrous strain B-276 and its alkene monooxygenase (AMO) gene cluster were the focus of our investigation. Firstly, 2 types of cultures of the R. rhodochrous strain B-276 were prepared, one of which was supplied with propene, which requires AMO genes for its assimilation, whereas the other was supplied with glucose as the sole energy source. The latter culture was anticipated to have a lower gene frequency of AMO genes because of their deletion during cultivation. We then conducted whole genome shotgun sequencing of the genomic DNA extracted from both cultures. Next, all sequence data were pooled and assembled into contiguous sequences (contigs). Finally, the abundance of each contig was quantified in order to detect contigs that were highly biased between the 2 cultures. We identified contigs that were overrepresented by 2 orders of magnitude in the AMO-required culture and successfully identified an AMO gene cluster among these contigs. We propose this procedure as an efficient method for the rapid detection and sequencing of deleted region, which contributes to identification of functional genes in rhodococci.

Mechanism of action of nitrous oxide gas applied as a polyploidizing agent during meiosis in lilies.

Nitrous oxide gas (N(2)O) can be used to produce polyploid plants, but the mechanism of action is unknown. The actin and microtubule cytoskeleton was observed in N(2)O-treated microsporocytes of Lilium spp 'Asiatic hybrid lilies' using fluorescence microscopy after staining with DAPI, FITC-conjugated tubulin antibody, and phalloidin-conjugated Alexa Fluor 546. Additionally, microsporocytes of L. longiflorum were observed with acetocarmine staining following N(2)O treatment. A typical metaphase I microtubule distribution was observed in control microsporocytes. After treatment with N(2)O for 24 h, microtubules were effectively depolymerized; this prevented chromosomes from moving to the poles, resulting in chromosome retention in the center of N(2)O-treated cells. Cell plate formation took place without delay, however, yielding one daughter cell with a diploid genome and another daughter without chromosomes. In addition, N(2)O treatment often induced micronuclei due to aberrant chromosome separation during cytokinesis. Actin filaments in microsporocytes are insensitive to N(2)O. These findings indicate that N(2)O mediates polyploidization by inhibiting microtubule polymerization, but not actin filament formation, during microsporocyte meiosis.