Japanese cedar (Cryptomeria japonica) is an important tree for Japanese forestry. Male-sterile marker development in Japanese cedar would facilitate selection of male-sterile plus trees, addressing the widespread social problem of pollinosis and facilitating the identification of heterozygotes, which are useful for breeding. This study used next-generation sequencing for single-nucleotide polymorphism discovery in libraries constructed from several organs, including male-sterile and male-fertile strobili. The single-nucleotide polymorphisms obtained were used to construct a high-density linkage map, which enabled identification of a locus on linkage group 9 strongly correlated with male-sterile trait. Expressed sequence tags corresponding to 11 marker loci from 5 isotigs were associated with this locus within 33.4-34.5 cM. These marker loci explained 100% of the phenotypic variation. Several homologs of these sequences are associated with male sterility in rice or Arabidopsis, including a pre-mRNA splicing factor, a DEAD-box protein, a glycosyl hydrolase, and a galactosyltransferase. These proteins are thus candidates for the causal male-sterile gene at the ms-1 locus. After we used a SNaPshot assay to develop markers for marker-assisted selection (MAS), we tested F2 progeny between male-sterile and wild-type plus trees to validate the markers and extrapolated the testing to a larger plus-tree population. We found that two developed from one of the candidates for the causal gene were suitable for MAS. More than half of the ESTs and SNPs we collected were new, enlarging the genomic basis for genetic research on Japanese cedar. We developed two SNP markers aimed at MAS that distinguished individuals carrying the male-sterile trait with 100% accuracy, as well as individuals heterozygous at the male-sterile locus, even outside the mapping population. These markers should enable practical MAS for conifer breeding.

Context: Clonal variation of flower production has been investigated thoroughly in coniferous tree species because of the importance of flowering in seed production from seed orchards. However, the inheritance of flower production by progeny test has not been previously studied well, so the heritability of male flower production of Cryptomeria japonica, the most important species for forestry and the main cause of pollinosis in Japan, was estimated to provide fundamental data for pollen control in seed orchards and plantation forests. Methods: Male flower production in C. japonica was investigated for three years using 20 full-sib families created by a diallel mating design with six parents using gibberellin treatment to promote flowering. Results: The narrow-sense individual tree heritability of male flower production in each year was estimated as ranging from 0.777 ± 0.339 to 1.050 ± 0.346 by sib analyses. The contribution of general combining ability to the total variance varied from 24.1 to 35.6 %. The highest contribution of specific combining ability was 1.42 %. By selecting the top 50 % of parental clones for reduced male flower production, the genetic gain in lowering male flower productivity was predicted as 46.1–53.0 %. Conclusion: Our results suggest that male flower production in C. japonica is under strong genetic control and that control of male flowering by breeding is possible with a well-designed program.

The plant hormone gibberellin (GA) is known to regulate elongating growth, seed germination, and the initiation of flower bud formation, and it has been postulated that GAs originally had functions in reproductive processes. Studies on the mechanism of induction of flowering by GA have been performed in Arabidopsis and other model plants. In coniferous trees, reproductive organ induction by GAs is known to occur, but there are few reports on the molecular mechanism in this system. To clarify the gene expression dynamics of the GA induction of the male strobilus in Cryptomeria japonica, we performed comprehensive gene expression analysis using a microarray. A GA-treated group and a nontreated group were allowed to set, and individual trees were sampled over a 6-week time course. A total of 881 genes exhibiting changed expression was identified. In the GA-treated group, genes related to ‘stress response’ and to ‘cell wall’ were initially enriched, and genes related to ‘transcription’ and ‘transcription factor activity’ were enriched at later stages. This analysis also clarified the dynamics of the expression of genes related to GA signaling transduction following GA treatment, permitting us to compare and contrast with the expression dynamics of genes implicated in signal transduction responses to other plant hormones. These results suggested that various plant hormones have complex influences on the male strobilus induction. Additionally, principal component analysis (PCA) using expression patterns of the genes that exhibited sequence similarity with flower bud or floral organ formation-related genes of Arabidopsis was performed. PCA suggested that gene expression leading to male strobilus formation in C. japonica became conspicuous within one week of GA treatment. Together, these findings help to clarify the evolution of the mechanism of induction of reproductive organs by GA.