Development of Genetic Markers for Sex and Individual Identification of the Japanese Giant Flying Squirrel (Petaurista leucogenys) by an Efficient Method Using High-Throughput DNA Sequencing.

DNA markers that detect differences in the number of microsatellite repeats can be highly effective for genotyping individuals that lack differences in external morphology. However, isolation of sequences with different microsatellite repeat numbers between individuals has been a time-consuming process in the development of DNA markers. Individual identification of Japanese giant flying squirrels (Petaurista leucogenys) has been challenging because this species is arboreal and nocturnal and exhibits little to no morphological variation between individuals. In this study, we developed DNA markers for sex and individual identification of this species by an efficient method using high-throughput DNA sequence data. Paired-end 5 Gb (2 × 250 bp) and 15 Gb (2 × 150 bp) genome sequences were determined from a female and a male Japanese giant flying squirrel, respectively. We searched SRY and XIST genes located on Y and X chromosomes, respectively, from high-throughput sequence data and designed primers to amplify these genes. Using these primer sets, we succeeded to identify the sex of individuals. In addition, we selected 12 loci containing microsatellites with different numbers of repeats between two individuals from the same data set, and designed primers to amplify these sequences. Twenty individuals from nine different locations were discriminated using these primer sets. Furthermore, both sex and microsatellite markers were amplified from DNA extracted non-invasively from single fecal pellet samples. Based on our results for flying squirrels, we expect our efficient method for developing non-invasive high-resolution individual- and sex-specific genotyping to be applicable to a diversity of mammalian species.

Automating measurements of fluorescent signals in freely moving plant leaf specimens.

Existing methods to quantify fluorescent signals are primarily limited to non-moving objects or tracking a limited number of cells. These techniques, however, are unsuitable for measuring fluorescent signals in time-lapse experiments using plant specimens that move naturally during a course of imaging. We developed an automated method to measure fluorescent signal intensities in transgenic Arabidopsis plants using a stereomicroscope with standard microscopy software. The features of our technique include: 1) recognizing the shape of plant specimens using autofluorescent signals; 2) merging targeted fluorescent signals to specimen outlines; 3) extracting signals within the shape of specimens from their background signals. Our method facilitates the measurement of fluorescent signals on freely moving plant leaves that are physically unrestrained. The method we developed addresses the challenge of recognizing plant shapes without relying on: a) manual definition which is prone to subjectivity and human error; b) introducing stable fluorescent markers to define plant shapes; c) recognizing plant shapes from bright field images which include a wide range of colors and background noise; d) unnecessarily stressing plants by immobilizing them; e) the use of multiple software packages or software development expertise.