Genetic characterization of the male-sterile Japanese cedar ‘Fukushima-funen 3’ reveals a naturally pyramided genotype and a novel MS2 allele, and leads to the development of marker-assisted selection for the MS2 locus

Pollen allergies are an increasingly serious public-health issue worldwide. Japanese cedar (Cryptomeria japonica D. Don, Cupressaceae) is an important forestry species in Japan that releases large amounts of airborne pollen in spring, causing severe allergic reactions. Planting male-sterile individuals is expected to reduce pollen dispersal effectively. ‘Fukushima-funen 3’ is a male-sterile individual whose causal gene has remained unidentified. Through test crossings, we determined that the gene responsible for male sterility in ‘Fukushima-funen 3’ is MS2, and that this individual is heterozygous at the MS1 locus. These findings were supported by microscopy observations of pollen development and genotyping using DNA markers targeting the causal mutations in MS1 and MS2 candidate genes. Sanger sequencing of the coding region of the MS2 candidate gene revealed that ‘Fukushima-funen 3’ has both a previously reported single-nucleotide substitution and a novel 8-bp deletion within the mutation site. We conducted marker-assisted selection on 1511 breeding materials collected from various regions of Japan using these two mutations as DNA markers, and identified eight breeding materials with male sterility alleles. Some of these individuals had male sterility alleles at multiple loci, implying their potential utility in gene-pyramiding-based breeding strategies. These findings will contribute to the genetic elucidation of male sterility in C. japonica and the development of trees with superior traits.