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 for the production of a pollen-free cultivar. In order to reduce the time and cost needed for the 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. The expected function of this gene, its two dysfunctional mutations, and genetic diversity were described recently in a related study.
Results We have developed PCR and LAMP markers to detect mutant alleles and to present experimental options depending on available laboratory equipment. At field stations, where PCR machines are not available, LAMP markers were developed. LAMP only needs heat-blocks or a water bath to perform the isothermal amplification and assay results can be easily seen by eye. Because the causative mutations were deletions, two kinds of PCR markers, amplified length polymorphism (ALP) and allele specific PCR (ASP) markers, were developed. These assays can be carried out by capillary or agarose gel electrophoresis.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- ALP
- amplified length polymorphism
- ARMS
- amplification refractory mutation system
- ASP
- allele specific PCR
- BIP
- backward inner primer
- bp
- base pair
- DNA
- deoxyribonucleic acid
- FIP
- forward inner primer
- Gbp
- giga base pair
- LAMP
- loop-mediated isothermal amplification
- PCR
- polymerase chain reaction
- PNA
- peptide nucleic acid
- RNA
- ribonucleic acid