Yumoto Keisuke, Kanbe Takashi, Saito Yoko, Kaneko Shingo, Tsuda Yoshiaki
Sugadaira Research Station, Mountain Science Center, University of Tsukuba, Nagano, Japan.
Systematic Entomology, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan.
Front Insect Sci. 2021 Jul 5;1:696886. doi: 10.3389/finsc.2021.696886. eCollection 2021.
Although insect exuviae-based genetics is challenging, it can be a valuable method for obtaining reliable DNA resources by non-invasive sampling. This approach is especially effective when the target species is endangered/endemic or when sampling the adult is difficult. One example is cicadas, which during molt leave their exoskeletons on tree trunks, making them easily collectable. While cicada exuviae-derived DNA has previously been employed for mitochondrial DNA sequencing, this study aimed to develop a reliable method for the PCR amplification of nuclear microsatellite loci from cicada exuviae derived DNA for application in molecular ecology, conservation and population genetics. Five different PCR amplification protocols were performed, and the fragment patterns compared with those obtained using DNA extracted from adult individuals. Moreover, the relationship between the freshness of the exuviae and genotyping success was evaluated. provided the best performance in the PCR amplification of DNA isolated from cicada exuviae and the electropherogram showed a clear fragment pattern that was equivalent to that obtained from the DNA extracted from the adult individual. This result suggests that cicada exuviae-derived DNA can be amplified by PCR and that multiple independent loci of nuclear DNA microsatellite markers can be easily genotyped. This study demonstrates that fresh cicada exuviae provide high quality DNA, which can be used for microsatellite genotyping. The methods developed in this study are applicable not only for cicada but other insect species for which exuviae are available. Thus, this study can make a significant contribution to insect sciences.
尽管基于昆虫蜕皮的遗传学研究具有挑战性，但它可以成为一种通过非侵入性采样获得可靠DNA资源的有价值方法。当目标物种处于濒危/特有状态或难以对成虫进行采样时，这种方法尤其有效。一个例子是蝉，它们在蜕皮时会将外骨骼留在树干上，便于收集。虽然之前已将蝉蜕皮衍生的DNA用于线粒体DNA测序，但本研究旨在开发一种可靠的方法，用于从蝉蜕皮衍生的DNA中PCR扩增核微卫星位点，以应用于分子生态学、保护遗传学和种群遗传学。进行了五种不同的PCR扩增方案，并将片段模式与使用从成虫个体提取的DNA获得的片段模式进行比较。此外，还评估了蜕皮的新鲜程度与基因分型成功率之间的关系。[某种方案]在从蝉蜕皮中分离的DNA的PCR扩增中表现最佳，电泳图谱显示出清晰的片段模式，与从成虫个体提取的DNA获得的片段模式相当。这一结果表明，蝉蜕皮衍生的DNA可以通过PCR扩增，并且核DNA微卫星标记的多个独立位点可以轻松进行基因分型。本研究表明，新鲜的蝉蜕皮可提供高质量的DNA，可用于微卫星基因分型。本研究中开发的方法不仅适用于蝉，也适用于有蜕皮可用的其他昆虫物种。因此，本研究可为昆虫科学做出重大贡献。
