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.

In the twentieth century, a substantial decline in Pinus thunbergii populations in Japan occurred due to the outbreak of pine wood nematode (PWN), Burshaphelencus xylophilus. A PWN-P. thunbergii resistant trees-breeding project was developed in the 1980s to provide reforestation materials to minimalize the pest damage within the population. Since climate change can also contribute to PWN outbreaks, an intensive reforestation plan instated without much consideration can impact on the genetic diversity of P. thunbergii populations. The usage and deployment of PWN-P. thunbergii resistant trees to a given site without genetic management can lead to a genetic disturbance. The Iki-no-Matsubara population was used as a model to design an approach for the deployment management. This research aimed to preserve local genetic diversity, genetic structure, and relatedness by developing a method for deploying Kyushu PWN-P. thunbergii resistant trees as reforestation-material plants into Iki-no-Matsubara. The local genotypes of the Iki-no-Matsubara population and the Kyushu PWN-P. thunbergii resistant trees were analyzed using six microsatellite markers. Genotype origins, relatedness, diversity, and structure of both were investigated and compared with the genetic results previously obtained for old populations of P. thunbergii throughout Japan. A sufficient number of Kyushu PWN-P. thunbergii resistant trees, as mother trees, within seed orchards and sufficient status number of the seedlings to deploy are needed when deploying the Kyushu PWN-P. thunbergii resistant trees as reforestation material planting into Iki-no-Matsubara population. This approach not only be used to preserve Iki-no-Matsubara population (genetic diversity, genetic structure, relatedness, and resilience of the forests) but can also be applied to minimize PWN damage. These results provide a baseline for further seed sourcing as well as develop genetic management strategies within P. thunbergii populations, including Kyushu PWN-P. thunbergii resistant trees.