Hasegawa Yoichi, Ueno Saneyoshi, Wei Fu-Jin, Matsumoto Asako, Ujino-Ihara Tokuko, Uchiyama Kentaro, Moriguchi Yoshinari, Kasahara Masahiro, Fujino Takeshi, Shigenobu Shuji, Yamaguchi Katsushi, Bino Takahiro, Hakamata Tetsuji
Forestry and Forest Products Research Institute, Forest Research and Management Organization, Tsukuba, Ibaraki, Japan.
Graduate School of Science and Technology, Niigata University, Niigata, Japan.
BMC Res Notes. 2020 Sep 29;13(1):457. doi: 10.1186/s13104-020-05296-8.
Due to the allergic nature of the pollen of Cryptomeria japonica, the most important Japanese forestry conifer, a pollen-free cultivar is preferred. Mutant trees detected in nature have been used to produce a pollen-free cultivar. In order to reduce the time and cost needed for production and breeding, we aimed to develop simple diagnostic molecular markers for mutant alleles of the causative gene MALE STERILITY 1 (MS1) in C. japonica to rapidly identify pollen-free mutants.
We developed PCR and LAMP markers to detect mutant alleles and to present experimental options depending on available laboratory equipment. LAMP markers were developed for field stations, where PCR machines are unavailable. The LAMP method only needs heat-blocks or a water bath to perform the isothermal amplification and assay results can be read by the naked eye. Because the causative mutations were deletions, we developed two kinds of PCR markers, amplified length polymorphism (ALP) and allele specific PCR (ASP) markers. These assays can be visualized using capillary or agarose gel electrophoresis.
由于日本最重要的林业针叶树日本柳杉的花粉具有过敏性，因此无花粉品种更受青睐。已利用在自然界中检测到的突变树来培育无花粉品种。为了减少生产和育种所需的时间和成本，我们旨在开发用于日本柳杉致病基因雄性不育1（MS1）突变等位基因的简单诊断分子标记，以快速鉴定无花粉突变体。
我们开发了PCR和LAMP标记来检测突变等位基因，并根据可用的实验室设备提供实验选择。为没有PCR仪的野外工作站开发了LAMP标记。LAMP方法仅需要加热块或水浴来进行等温扩增，并且检测结果可以肉眼读取。由于致病突变是缺失，我们开发了两种PCR标记，即扩增片段长度多态性（ALP）和等位基因特异性PCR（ASP）标记。这些检测可以使用毛细管或琼脂糖凝胶电泳进行可视化。
