Development of polymorphic microsatellite loci in Odontobutis obscura using Illumina paired-end sequencing and the test of cross-species amplification.

Odontobutis obscura is a bottom-dwelling freshwater fish native to East Asia. Its range encompasses southwest China, western Japan, and Geoje Island in South Korea. Despite its widespread range in China and Japan, only a small and spatially isolated population is found in South Korea. We developed a total of 23 novel and polymorphic microsatellite loci of O. obscura using Illumina paired-end shotgun sequencing and characterized them using 80 Japanese and Korean samples. An extensive genetic polymorphism was detected at these 23 loci, with the observed number of alleles at a locus ranging from 2 to 15 and expected and observed heterozygosities ranging from 0 to 0.656 and 0 to 0.547, respectively. Korean O. obscura exhibited a much lower level of genetic variability than the Japanese population did, probably as a result of long-term isolation combined with historical bottlenecks. The Japanese and Korean populations showed a high level of genetic differentiation with FST = 0.700 and RST = 0.913. Many of our primer sets were successfully transferable to congeneric O. interrupta and O. platycephala, which exhibited even greater polymorphism than Korean O. obscura. In conclusion, our study showed that these 23 microsatellite markers are useful for understanding the conservation biology and population genetic structure of O. obscura and other congeneric species.

Development of novel microsatellite markers for conservation genetic studies of Vulpes vulpes (Canidae) by using next-generation sequencing method.

The red fox, Vulpes vulpes (Canidae), is the most widely distributed terrestrial carnivore worldwide, but this species is classified as endangered in Korea. In this study, we developed 25 polymorphic microsatellite markers that included 3-13 (mean = 6.32) alleles per locus using 22 red fox individuals. The most polymorphic locus was FR(59)TG (13 alleles) and the least polymorphic loci were FR(70)TG and FR(182)AG (3 alleles each). No significant deviation from Hardy-Weinberg equilibrium (P < 0.05) was observed for the 25 markers. Observed (HO) and expected (HE) heterozygosity varied from 0.182 to 1.000 and from 0.175 to 0.929, respectively. These newly developed microsatellite markers will be useful for investigating the genetic diversity and population genetic structure of V. vulpes and will aid in developing conservation strategies for this species.

Next-generation sequencing identification and characterization of microsatellite markers in Aconitum austrokoreense Koidz., an endemic and endangered medicinal plant of Korea.

We used next-generation sequencing to develop 9 novel microsatellite markers in Aconitum austrokoreense, an endemic and endangered medicinal plant in Korea. Owing to its very limited distribution, over-harvesting for traditional medicinal purposes, and habitat loss, the natural populations are dramatically declining in Korea. All novel microsatellite markers were successfully genotyped using 64 samples from two populations (Mt. Choejeong, Gyeongsangbuk-do and Ungseokbong, Gyeongsangnam-do) of Gyeongsang Province. The number of alleles ranged from 2 to 7 per locus in each population. Observed and expected heterozygosities ranged from 0.031 to 0.938 and from 0.031 to 0.697, respectively. The novel markers will be valuable tools for assessing the genetic diversity of A. austrokoreense and for germplasm conservation of this endangered species.

Identification of RNA editing sites in cotton (Gossypium hirsutum) chloroplasts and editing events that affect secondary and three-dimensional protein structures.

RNA editing can alter individual nucleotides in primary transcripts, which can cause the amino acids encoded by edited RNA to deviate from the ones predicted from the DNA template. We investigated RNA editing sites of protein-coding genes from the chloroplast genome of cotton. Fifty-four editing sites were identified in 27 transcripts, which is the highest editing frequency found until now in angiosperms. All these editing sites were C-to-U conversion, biased toward ndh genes and U_A context. Examining published editotypes in various angiosperms, we found that RNA editing mostly converts amino acid from hydrophilic to hydrophobic and restores evolutionary conserved amino acids. Using bioinformatics to analyze the effect of editing events on protein secondary and three-dimensional structures, we found that 21 editing sites can affect protein secondary structures and seven editing sites can alter three-dimensional protein structures. These results imply that 24 editing sites in cotton chloroplast transcripts may play an important role in their protein structures and functions.