Kyoko Sugai

Rapid development of microsatellite markers for Pandanus boninensis (Pandanaceae) by pyrosequencing technology.

PREMISE OF THE STUDY To facilitate rapid development of microsatellite or simple sequence repeat (SSR) markers, an expressed sequence tags (EST) database was constructed for Pandanus boninensis, an evergreen tree endemic to the Bonin Islands, using pyrosequencing technology. METHODS AND RESULTS We designed primers for 340 EST-SSRs identified from 109620 pyrosequencing reads, 48 of which were tested for PCR amplification. Thirty-four primers provided clear amplification, and 26 of those 34 displayed clear polymorphic patterns in sampled populations, with mean expected heterozygosity at the amplified loci ranging from 0.022 to 0.742 (average 0.262). CONCLUSIONS The developed markers are promising tools for future genetic studies of P. boninensis and related species.

Microsatellite markers derived from Calophyllum inophyllum (Clusiaceae) expressed sequence tags

PREMISE OF THE STUDY Robust markers are required (inter alia) for assessing origins of Calophyllum inophyllum populations on the Bonin Islands, Japan. Therefore, informative expressed sequence tag (EST)-based microsatellite or simple sequence repeat (SSRs) markers in the species were sought. METHODS AND RESULTS Using 135378 ESTs derived from de novo pyrosequencing, primers for 475 EST-SSRs were developed, 48 of which were tested for PCR amplification. Thirty-six of the 48 primers showed clear amplification, with 23 displaying polymorphism in sampled populations. Expected heterozygosity in the samples from the Bonin Islands and Ryukyu Islands populations ranged from 0.041 to 0.697 and from 0.041 to 0.773, respectively. CONCLUSIONS As EST-SSRs are potentially tightly linked with functional genes, and reportedly more transferable to related species than anonymous genomic SSRs, the developed primers have utility for future studies of the origins, genetic structure, and conservation of C. inophyllum and related species.

Isolation and characterization of EST-SSR markers in Schima mertensiana (Theaceae) using pyrosequencing technology.

PREMISE OF THE STUDY Microsatellite or simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) in Schima mertensiana, an evergreen tree that is endemic to the Bonin Islands, were developed to facilitate evaluation of the speciess population genetic structure. METHODS AND RESULTS Using 149831 ESTs derived by pyrosequencing S. mertensiana complementary DNA (cDNA), 683 EST-SSRs were developed, 48 of which were tested for amplification. Thirty of the 48 showed clear amplification and detected polymorphism in sampled populations, with mean expected heterozygosity at the amplified loci ranging from 0.045 to 0.874 (average 0.545). CONCLUSIONS The markers developed in this study can be used for future studies of the genetic structure of S. mertensiana and related species.

Isolation and characterization of Ligustrum micranthum (Oleaceae) microsatellite loci using paired‐end Illumina reads

Twenty-six microsatellite loci were developed and characterized for Ligustrum micranthum, a species endemic to the Ogasawara Islands, Japan. The genetic structure of this species must be clarified in order to restore the islands ecosystem. A total of 8511 primer pairs were designed from de novo sequencing. Of the 48 primer pairs selected, amplification and polymorphisms were tested using one population each from the Chichijima and Hahajima Islands of the Ogasawara Islands. Twenty-six microsatellite loci were successfully amplified and the number of alleles for these loci ranged from five to 31 per locus, and the mean expected heterozygosities were 0.858 and 0.849, respectively. No significant deviation from the Hardy–Weinberg equilibrium was observed in either population, and no significant linkage disequilibrium was detected between any locus pair. The microsatellite loci reported in this study can be used in future studies to evaluate the genetic structure and mating system of L. micranthum.

Novel microsatellite markers for Distylium lepidotum (Hamamelidaceae) endemic to the Ogasawara Islands

BackgroundDistylium lepidotum is a small tree endemic to the Ogasawara Islands located in the northwestern Pacific Ocean. This species is a sole food for an endemic locust, Boninoxya anijimensis. Here, we developed microsatellite markers to investigate genetic diversity and genetic structure and to avoid a genetic disturbance after transplantation to restore the Ogasawara Islands ecosystem.ResultsMicrosatellite markers with perfect dinucleotide repeats were developed using the next-generation sequencing Illumina MiSeq Desktop Sequencer. Thirty-two primer pairs were characterized in two D. lepidotum populations on Chichijima and Hahajima Islands of the Ogasawara Islands. The number of alleles for the markers ranged from three to 23 per locus in the two populations. Expected heterozygosity per locus in each population ranged from 0.156 to 0.940 and 0.368 to 0.845, respectively.ConclusionsThese microsatellite markers will be useful for future population genetics studies of D. lepidotum and provide a basis for conservation management of the Ogasawara Islands.

Development of EST-SSR markers for Elaeocarpus photiniifolia (Elaeocarpaceae), an endemic taxon of the Bonin Islands

PREMISE OF THE STUDY Expressed sequence tag (EST)-derived microsatellite markers were developed for Elaeocarpus photiniifolia, an endemic taxon of the Bonin Islands. METHODS AND RESULTS Initially, a complementary DNA (cDNA) library was constructed by de novo pyrosequencing of total RNA extracted from a seedling. A total of 267 primer pairs were designed from the library. Of the 48 tested loci, 25 loci were polymorphic among 41 individuals representing the entire geographical range of the species, with the number of alleles per locus and expected heterozygosity ranging from two to 14 and 0.09 to 0.86, respectively. Most loci were transferable to a related species, E. sylvestris. CONCLUSIONS The developed markers will be useful for evaluating the genetic structure of E. photiniifolia.

Genetic variation of pantropical Terminalia catappa plants with sea-drifted seeds in the Bonin Islands: suggestions for transplantation guidelines

The Bonin Islands are endowed with endemic species. However, these species are at risk of extinction because of the exuberance of invasive alien plants. Therefore, native plant species should be revegetated after eradicating alien plants. We investigated the genetic variation of Terminalia catappa populations in the Bonin Islands by using nuclear (n) microsatellites (simple sequence repeats [SSRs]) and chloroplast (cp) DNA. No significant differences were observed in the genetic diversity of nSSRs among 22 populations. However, recent bottlenecks were detected in three populations on the Chichijima Island group. nSSR variation and cpDNA haplotypes suggested the presence of two genetically distinct groups in the Mukojima and Chichijima Island groups and the Hahajima Island group. A similar genetic structure was observed in plants and animals in the Bonin Islands. Populations on the three islands, which were separated from other islands in each island group when the water depth was 50-m lower than the present level, were dominated by unique nSSRs clusters, suggesting that historical changes in island connections during the Pleistocene era affected genetic substructuring. These results suggested that different factors contributed to the genetic structure of T. catappa on different geographic scales. At the whole-island level, the genetic structure was determined by long-distance seed dispersal by ocean currents. At the island-group level, the genetic structure was determined by historical changes in island connections caused by changes in the sea level due to glacial–interglacial transition. These findings would help in establishing transplantation zone borders for revegetating T. catappa on the Bonin Islands.

Development and evaluation of microsatellite loci for Gynochthodes boninensis (Rubiaceae), a woody climbing plant endemic to the Bonin (Ogasawara) Islands, Japan

Microsatellite loci were developed for Gynochthodes boninensis, an endemic climbing plant in the Bonin Islands. Using a Roche 454 GS Junior next-generation sequencer, 158 microsatellite loci were designed. Of the 48 microsatellite loci tested, 37 were successfully amplified and 25 were polymorphic in two populations of G. boninensis. For the 25 polymorphic loci, the mean expected heterozygosities per locus were 0.303 in the Chichijima Island population and 0.310 in the Hahajima Island population, respectively. There was no evidence of linkage disequilibrium in either population, but one locus showed significant deviation from Hardy–Weinberg equilibrium in one population. The microsatellite loci developed in this study will be useful for future studies of population genetics of G. boninensis. In particular, because this species is androdioecious (males and hermaphrodites coexist), characterizing the species gene flow is crucial to understanding the evolution and maintenance of this rare sexual system.

Development of SSR markers for Psychotria homalosperma (Rubiaceae) and cross-amplification in four other species.

Premise of the study: Twenty-six microsatellite (simple sequence repeat [SSR]) markers were characterized in Psychotria homalosperma (Rubiaceae), an endemic evergreen tree in the Bonin Islands, Japan, to investigate the genetic structure and gene flow of the species. Methods and Results: Using next-generation sequencing, we developed 26 SSR markers for P. homalosperma with perfect motifs from di- to pentanucleotide repeats. Of these, the Chichijima and Hahajima island populations of P. homalosperma had mean allele numbers of 6.50 and 6.81, respectively. The mean expected heterozygosities were 0.578 and 0.606, respectively. In addition, 10 and eight of these markers were successfully amplified for P. boninensis and P. serpens, respectively, occurring in the same or adjacent areas. Conclusions: The SSR markers developed in this study will be useful for future studies concerning the population genetics of P. homalosperma and will facilitate the development of a conservation strategy.