Development and characterization of nuclear microsatellite markers in Aphananthe aspera (Thunb.) Planch. (Cannabaceae)

Nuclear microsatellite markers were developed for Aphananthe aspera (Thunb.) Planch. (Cannabaceae), a deciduous canopy tree species distributed in East Asia, to evaluate the genetic diversity and genetic structure of A. aspera populations in remnant forest fragments in urbanized areas of Japan. A total of 94 primer pairs were designed based on genomic sequence data. Of the 25 primer pairs which showed clear microsatellite peaks, 20 pairs showed allelic polymorphisms in 57 individuals collected from two distant populations. The length of PCR products ranged from 120 to 482 bp, and expected heterozygosity for the 20 microsatellite markers ranged from 0.017 to 0.768. These newly developed simple sequence repeat markers will be used in population genetic studies of A. aspera to evaluate genetic diversity and the extent of genetic isolation of the fragmented populations in urban areas

Application of a Simplified Method of Chloroplast Enrichment to Small Amounts of Tissue for Chloroplast Genome Sequencing

Premise of the study: High-throughput sequencing of genomic DNA can recover complete chloroplast genome sequences, but the sequence data are usually dominated by sequences from nuclear/mitochondrial genomes. To overcome this deficiency, a simple enrichment method for chloroplast DNA from small amounts of plant tissue was tested for eight plant species including a gymnosperm and various angiosperms. Methods: Chloroplasts were enriched using a high-salt isolation buffer without any step gradient procedures, and enriched chloroplast DNA was sequenced by multiplexed high-throughput sequencing. Results: Using this simple method, significant enrichment of chloroplast DNA-derived reads was attained, allowing deep sequencing of chloroplast genomes. As an example, the chloroplast genome of the conifer Callitris sulcata was assembled, from which polymorphic microsatellite loci were isolated successfully. Discussion: This chloroplast enrichment method from small amounts of plant tissue will be particularly useful for studies that use sequencers with relatively small throughput and that cannot use large amounts of tissue (e.g., for endangered species)