Forest trees have ecological and economic importance, and Japanese cedar has highly valued wood attributes. Thus, studies of molecular aspects of wood formation offer practical information that may be used for screening and forward genetics approaches to improving wood quality.

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.

Background: Pine wilt disease (PWD), which is caused by the pine wood nematode (PWN)Bursaphelenchus xylophilus, is currently the greatest threat to pine forests in Europe and East Asian countries including Japan. Constructing a detailed linkage map of DNA markers and identifying PWD resistance genes/loci lead to improved resistance inPinus thunbergii, as well as otherPinusspecies that are also susceptible to PWD.Results: A total F₁mapping population of 188 individuals derived from a cross between the PWD-resistantP. thunbergiivarieties ‘Tanabe 54’ (resistant rank 2 to PWD) and ‘Tosashimizu 63’ (resistant rank 4 to PWD) was inoculated with PWN, and was evaluated for disease symptoms. To perform linkage analysis for PWN resistance, a set of three maps was constructed; two parental maps generated using the integrated two-way pseudo-testcross method, and a consensus map with population-type cross-pollination. The linkage map of ‘Tanabe 54’ consisted of 167 loci, and covered 14 linkage groups (LGs), with a total genetic distance of 1214.6 cM. The linkage map of ‘Tosashimizu 63’ consisted of 252 loci, and covered 14 LGs, with a total genetic distance of 1422.1 cM. The integrated consensus map comprised 12 LGs with the basic chromosome number ofP. thunbergii, and a total genetic distance of 1403.6 cM. Results from quantitative trait loci (QTL) analysis using phenotype data and linkage maps indicated that PWN resistance is controlled by a single dominant allele, which was derived from the ‘Tanabe 54’ female parent. This major QTL was located on linkage group 3 and was designatedPWD1forPINE WILT DISEASE 1.Conclusions: ThePWD1locus is a major resistance QTL located on thePinusconsensus LG03 that acts in a dominant manner to confer pine wood nematode resistance. Information from the present study will be useful forP. thunbergiibreeding programs to improve resistance to PWD, and also to help identify susceptibility genes inPinusspecies.

Adventitious root formation is an essential physiological process for successful propagation of cuttings in various plant species. Because coniferous species are highly heterozygous, propagation of cuttings is of great practical use in breeding. Although various factors influence adventitious root formation, little is known of the associated regulatory mechanisms. Whereas adventitious roots generally form from the base of cuttings, this process is accompanied by physiological changes in leaves, which supply assimilates and metabolites. Herein, we present microarray analyses of transcriptome dynamics during adventitious root formation in whole cuttings in the coniferous species, Cryptomeria japonica. Temporal patterns of gene expression were determined in the base, the middle, and needles of cuttings at eight time points during adventitious root formation. Global gene expression at the base had diverged from that in the middle by 3-h post-insertion, and changed little in the subsequent 3-days post-insertion, and global gene expression in needles altered characteristically at 3- and 6-weeks post-insertion. In Gene Ontology enrichment analysis of major gene clusters based on hierarchical clustering, the expression profiles of genes related to carbohydrates, plant hormones, and other categories indicated multiple biological changes that were involved in adventitious root formation. The present comprehensive transcriptome analyses indicate major transcriptional turning and contribute to the understanding of the biological processes and molecular factors that influence adventitious root formation in C. japonica.

Along with progress in sequencing technology and accumulating knowledge of genome and gene sequences, molecular breeding techniques have been developed for predicting the genetic potential of individual genotypes and for selecting superior individuals. For Japanese cedar (Cryptomeria japonica (L.f.) D.Don), which is the most common coniferous species in Japanese forestry, we constructed a custom primer panel for target amplicon sequencing in order to simultaneously determine 3034 informative single nucleotide polymorphisms (SNPs). We performed primary evaluation of the custom primer panel with actual sequencing and in silico PCR. Genotyped SNPs had a distribution over almost the entire region of the C. japonica linkage map and verified the high reproducibility of genotype calls compared to SNPs obtained by genotyping arrays. Genotyping was performed for 576 individuals of the F₁ population, and genomic prediction models were constructed for growth and wood property-related traits using the genotypes. Amplicon sequencing with the custom primer panel enables efficient obtaining genotype data in order to perform genomic prediction, manage clones, and advance forest tree breeding.