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The heartwood color of a major plantation tree Cryptomeria japonica shows high variability among clones and cultivars, and brighter heartwood has higher value in the usage of non-laminated wood such as in traditional construction, which makes heartwood color an important trait in breeding of this species. However, the genetic basis of the interactions between genetics and the environment on heartwood color has been understudied while these are necessary for effective breeding programs in multiple environmental condition. The objectives of the present study were to evaluate the effects of genetics and environments on heartwood color and how they interact in contrasting environments, and to identify genomic regions controlling heartwood color in C. japonica across multiple environments. Heartwood color in terms of L*a*b* color space and spectral reflectance was measured in common gardens established in three contrasting sites. Quantitative trait loci (QTL) that affect heartwood color were identified using previously constructed highly saturated linkage maps. Results found that heartwood color was largely genetically controlled, and genotype-by-environment interaction explained one-third of the total genetic variance of heartwood color. The effect of the environment was small compared to the effect of genetics, whereas environmental effects largely varied among heartwood color traits. QTL analysis identified a large number of QTLs with small to moderate effects (phenotypic variation explained of 6.6% on average). Some of these QTLs were stably expressed in multiple environments or had pleiotropic effects on heartwood color and moisture content. These results indicated that genetic variation in phenotypic plasticity plays an important role in regulating heartwood color and that the identified QTLs would maximize the breeding efficiency of heartwood color in C. japonica in heterogeneous environments.

Pollinosis caused by Japanese cedar (Cryptomeria japonica) is a widespread social problem in Japan. To date, 23 male-sterile C. japonica trees have been selected in Japan to address pollinosis, from which four male-sterility loci (MS1, MS2, MS3, and MS4) have been identified from test crossing results. For efficient breeding of male-sterile C. japonica trees, more male-sterile individuals and individuals heterozygous for male-sterile genes are required. Therefore, we aimed to develop DNA markers for marker-assisted selection of four types of male-sterile genes from populations without a family structure. First, for four families exhibiting segregation of each male-sterile locus (MS1, MS2, MS3, and MS4), genome-wide single-nucleotide polymorphism and insertion/deletion (indel) genotyping was performed using the Axiom myDesign Targeted Genotyping Array method. Four high-density linkage maps for mapping the MS1, MS2, MS3, and MS4 families were constructed, which included 4923, 1722, 1896, and 2247 markers, respectively. In these maps, 15, 4, 2, and 2 markers were located 0.0, 3.3, 1.1, and 0.0 cM from the MS1, MS2, MS3, and MS4 loci, respectively. Second, for the markers located 0.0 cM from a male-sterile locus (i.e., MS1 and MS4), to clarify the most tightly linked markers, we calculated the prediction rate of male-sterile gene genotypes from marker genotypes for 78 trees. The markers with the highest prediction rates were AX-174127446 (0.95) for MS1 and AX-174121522 (1.00) for MS4. The AX-174121522 marker was considered to be suitable for selecting trees homozygous or heterozygous for the MS4 gene from plus-trees without a pollination test, which requires a large amount of time and effort. The nearest markers to the male-sterile loci found in this study may facilitate the isolation of male-sterile genes in C. japonica via combination with the draft genomic sequence that is currently being collated.

Deep sequencing of small RNAs (sRNAs) in developing male strobili of second-generation offspring originating from a nuclear genic male sterile tree of Cryptomeria japonica were performed to characterize sRNA populations in the male strobili at early pollen developmental stages. Comparing to sequences of microRNA (miRNA) families of plant species and sRNAs expressed in the reproductive organs of representative vascular plants, 37 conserved miRNA families were detected, of which eight were ubiquitously expressed in the reproductive organs of land plant species. In contrast, miR1083 was common in male reproductive organs of gymnosperm species but absent in angiosperm species. In addition to conserved miRNAs, 199 novel miRNAs candidates were predicted. The expression patterns of the obtained sRNAs were further investigated to detect the differentially expressed (DE) sRNAs between genic male sterile and fertile individuals. A total of 969 DE sRNAs were obtained and only three known miRNA families were included among them. These results suggest that both conserved and species-specific sRNAs contribute to the development of male strobili in C. japonica.

Genome-wide association studies (GWAS) are an alternative to bi-parental QTL mapping in long-lived perennials. In the present study, we examined the potential of GWAS in conifers using 367 unrelated plus trees of Cryptomeria japonica D. Don, which is the most widely planted and commercially important tree species in Japan, and tried to detect significant associations between wood property traits and quantity of male strobili on the one hand, and 1,032 single nucleotide polymorphisms (SNPs) assigned to 1,032 genes on the other. Association analysis was performed with the mixed linear model taking into account kinship relationships and subpopulation structure. In total, 6 SNPs were found to have significant associations with the variations in phenotype. These SNPs were not associated with the positions of known genes and QTLs that have been reported to date, thus they may identify novel QTLs. These 6 SNPs were all found in sequences showing similarities with known genes, although further analysis is required to dissect the ways in which they affect wood property traits and abundance of male strobili. These presumptive QTL loci provide opportunities for improvement of C. japonica, based on a marker approach. The results suggest that GWAS has potential for use in future breeding programs in C. japonica.