SOKENDAI (The Graduate University for Advanced Studies), Department of Evolutionary Studies of Biosystems, Hayama, Kanagawa 240-0193, Japan,
Tama Forest Science Garden, Forestry and Forest Products Research Institute, Hachioji City, Tokyo 193-0843, Japan.
Zoolog Sci. 2023 Feb;40(1):24-31. doi: 10.2108/zs220045.
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 () 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.
可以检测微卫星重复次数差异的 DNA 标记物对于对外部形态没有差异的个体进行基因分型非常有效。然而，在开发 DNA 标记物时，分离个体之间具有不同微卫星重复数的序列一直是一个耗时的过程。由于日本巨型飞鼠是树栖和夜行性的，个体之间几乎没有形态变异，因此对其进行个体识别一直具有挑战性。在这项研究中，我们开发了用于该物种的性和个体识别的 DNA 标记物，该方法使用高通量 DNA 序列数据，效率很高。从一只雌性和一只雄性日本巨型飞鼠中分别确定了 5 Gb（2×250 bp）和 15 Gb（2×150 bp）的基因组序列。我们从高通量序列数据中分别搜索了位于 Y 和 X 染色体上的 SRY 和 XIST 基因，并设计了用于扩增这些基因的引物。使用这些引物组，我们成功地鉴定了个体的性别。此外，我们从同一数据集的两个个体之间具有不同重复数的 12 个微卫星基因座中选择了 12 个微卫星基因座，并设计了用于扩增这些序列的引物。使用这些引物组可以区分来自九个不同地点的二十个个体。此外，从单个粪便样本中提取的 DNA 也可扩增出这些微卫星标记和性染色体标记。基于我们对飞鼠的研究结果，我们预计我们开发非侵入性高分辨率个体和性别特异性基因分型的有效方法将适用于多种哺乳动物物种。
