Development of mitochondrial DNA cytochrome c oxidase subunit I primer sets to construct DNA barcoding library using next-generation sequencing.

Insects are one of the most diverse eukaryotic groups on the planet, with one million or more species present, including those yet undescribed. The DNA barcoding system has been developed, which has aided in the identification of cryptic species and undescribed species. The mitochondrial cytochrome c oxidase I region (mtDNA COI) has been utilised for the barcoding analysis of insect taxa. Thereafter, next-generation sequencing (NGS) technology has been developed, allowing for rapid acquisition of massive amounts of sequence data for genetic analyses. Although NGS-based PCR primers designed to amplify the mtDNA COI region have been developed, their target regions were only a part of COI region and/or there were taxonomic bias for PCR amplification. As the mtDNA COI region is a traditional DNA marker for the DNA barcoding system, modified primers for this region would greatly contribute to taxonomic studies. In this study, we redesigned previously developed PCR primer sets that targetted the mtDNA COI barcoding region to improve amplification efficiency and to enable us to conduct sequencing analysis on NGS. As a result, the redesigned primer sets achieved a high success rate (> 85%) for species examined in this study, covering four insect orders (Coleoptera, Lepidoptera, Orthoptera and Odonata). Thus, by combining the primers with developed primer sets for 12S or 16S rRNA regions, we can conduct more detailed taxonomic, phylogeographic and conservation genetic studies using NGS.

Flora of herbaceous and arboreous plants in Komaba Campus of the University of Tokyo, Japan.

BACKGROUND: Recent studies revealed that green spaces in urban areas are critical for conservation of native biodiversity and that assessment of the present flora of green spaces in urban areas is critical for protection of the native biodiversity. The Komaba Campus of the University of Tokyo embraces a relevant green area, located in a highly urbanised area in Tokyo Metropolis (35.66 N 139.68 E, Japan). The total area of this Campus is 25.4 ha, from which, 4.5 ha are covered by vegetation. Although intense urbanisation can be observed around the Campus, new insect species had been reported for the Campus area, suggesting that the biodiversity on the Campus still demands some attention. Differently from fauna surveys, no flora survey has been done for more than 30 years on the Campus. In this study, we have extensively surveyed the plants diversity on the Komaba Campus of the University of Tokyo, aiming for an update of the plants list on this green urban area in Tokyo. NEW INFORMATION: The survey covered all herbaceous and arboreous plants growing wild on the Campus. Garden plants were excluded in this survey because these plants were supposed to be cultivated. The final dataset contained, in total, 324 taxa, from which 234 were herbaceous plants and 90 were arboreous plants. The top three taxa are as follows: Poaceae (38 taxa), Asteraceae (34 taxa) and Rosaceae (14 taxa), respectively. This is the first update to the Flora of the Komaba Campus of the University of Tokyo in 30 years and represent an important contribution to conservation of native species in the Tokyo metropolis.

Development and characterization of nuclear microsatellite markers to reveal the neutral demographic background of flower color polymorphism in Geranium thunbergii (Geraniaceae).

Nuclear microsatellite markers were developed for Geranium thunbergii, an herbaceous plant characterized by petal color polymorphism. Utilizing RNA sequencing data obtained by next-generation sequencing techniques, we developed and characterized 19 polymorphic microsatellite markers with two to 12 alleles in the nuclear genome. These markers will be used to reveal the genetic structure and demographic history of G. thunbergii in the Japanese archipelago, which will elucidate the genetic background of flower color polymorphism among populations.

Development of nuclear and chloroplast microsatellite markers for the endangered conifer Callitris sulcata (Cupressaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for Callitris sulcata (Cupressaceae), an endangered conifer species in New Caledonia. METHODS AND RESULTS: Using sequencing by synthesis (SBS) of an RNA-Seq library, 15 polymorphic nuclear and chloroplast microsatellite markers were developed. When evaluated with 48 individuals, these markers showed genetic variations ranging from two to 15 alleles and expected heterozygosity ranging from 0 to 0.881. CONCLUSIONS: These markers will be useful for examining the genetic diversity and structure of remaining wild populations and improving the genetic status of ex situ populations.