Development of diagnostic PCR and LAMP markers for MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don
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RESEARCH NOTE
Development of diagnostic PCR and LAMP markers for MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don Yoichi Hasegawa1 , Saneyoshi Ueno1*, Fu‑Jin Wei1, Asako Matsumoto1, Tokuko Ujino‑Ihara1, Kentaro Uchiyama1, Yoshinari Moriguchi2, Masahiro Kasahara3, Takeshi Fujino3, Shuji Shigenobu4, Katsushi Yamaguchi4, Takahiro Bino4 and Tetsuji Hakamata5
Abstract Objective: 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. Results: 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. Keywords: ALP, ASP, LAMP, Molecular markers, Variant detection, Cryptomeria japonica, Sugi Introduction Molecular markers allow for the selection of specific phenotypes, once the genetic linkage of markers with associated genotypes and candidate gene(s) are identified. Due to the large body mass of tree species, especially conifers, maker-assisted selection with a candidate gene at the early life stage (e.g., seedling) is helpful in reducing the cost and time of breeding. Sugi (Cryptomeria japonica D. Don) is the most important forestry conifer in Japan, it occupies about 40% of artificial forests. Due to its fast growth and straight bowl, sugi timber has *Correspondence: [email protected] 1 Forestry and Forest Products Research Institute, Forest Research and Management Organization, Tsukuba, Ibaraki, Japan Full list of author information is available at the end of the article
been used for building materials and for daily life consumables, such as chopsticks and bowls. Recently, however, the pollen of sugi has caused seasonal allergies, which affects approximately 25% of Japanese citizens [1]. Therefore, breeding efforts are currently focused on producing a sterile pollen cultivar [2]. A candidate gene for male sterility, MALE STERILITY 1 (MS1), has been identified by transcriptome analysis and linkage mapping [3–5]. Sequencing analysis of the gene (CJt020762: 2,479 bp) from 83 samples identified 49 haplotypes with observed (HO) and expected (HE) heterozygosity of 0.892 and 0.920, respectively (Additional file 1), along
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