A simple allele-specific PCR marker for identifying male-sterile trees: Towards DNA marker-assisted selection in the Cryptomeria japonica breeding program

The number of people in Japan suffering from Cryptomeria japonica pollinosis has risen considerably since the 1970s as the area planted with this species has increased. In order to reduce the amount of pollen dispersed, breeding programs using trees with male-sterile genes have been implemented. We have constructed partial linkage maps surrounding a male sterility gene (ms-1) in four families of C. japonica to facilitate this process. The marker most closely linked to ms-1 was different in the four mapping families: gSNP00438, gSNP01452, estSNP00083, and estSNP01228 in the TO13S family (3.1 cM from ms-1); gSNP05835 and gSNP06239 in the S3T67 family (2.0 cM from ms-1); gSNP05835 in the F1N4 family (1.5 cM from ms-1); and gSNP06239 in the T5 family (4.2 cM from ms-1). This is probably mainly due to genetic differences between the parents used to produce the mapping families. However, in all four families, the accuracy with which male-sterile trees could be identified using the closest markers was more than 96.0 %. These results suggested that marker-assisted selection of male-sterile trees within a given family is feasible using the closest flanking markers to the ms-1 locus. We also developed an allele-specific PCR marker for identifying male-sterile trees in the TO13S family from which male-sterile seedlings are produced. Allele-specific PCR using three primer combinations produced two clear fragments, which could be easily separated by agarose gel electrophoresis: one fragment with a molecular weight of 410 bp, which was present in all samples and could thus be used as a positive control, and another of lower molecular weight (196 bp), which was specific for male-sterile trees. This marker makes it possible to carry out a simple and economical PCR assay for the detection of the SNP linked to the target gene without the need to use fluorescent labels. This study shows how a simple allele-specific PCR marker for an important major gene in a forest tree species can be developed using information from a high-density linkage map. In addition, our results will facilitate the first application of MAS (marker assisted selection) in conifers because the male sterility in C. japonica has several advantages and may be one of the best examples for MAS in conifers.