High-density linkage maps facilitate not only the understanding of genome structure and comparative genomic studies, but also quantitative trait loci (QTL) mapping and the construction of partial linkage maps around target loci to develop markers for marker-assisted selection (MAS). MAS is an effective method for accelerating the screening of target genes using tightly-linked molecular markers. Effective MAS for QTLs or specific genes has been reported in crops such as barley , rice  and tomato . However, it is difficult to employ MAS in conifers, primarily because their genomes are very big and complex and are largely uncharacterized. Indeed, very few DNA markers linked to major genes have been reported in conifers; the only ones of note are some dominant markers linked to a major dominant resistance gene in Pinus lambertiana [4, 5], P. monticola , P. taeda , and P. thunbergii [8, 9].
Sugi (Cryptomeria japonica D. Don) is an allogamous, diploid, wind-pollinated conifer species with a haploid chromosome number (n) of 11 (2n = 22). Using flow cytometry, the DNA content of its haploid cells was estimated to be 11.045 pg/C , which corresponds to a haploid genome size of 10.8 Gb as calculated using the following expression: genome size (bp) = 0.978 × 109 × DNA content (pg) . C. japonica is frequently used for commercial afforestation in Japan, and about 45% of all the man-made forests of Japan are composed of this species. However, since the 1970s, the incidence of C. japonica pollinosis in Japan has risen in line with the increasing number of man-made C. japonica forests . Today, C. japonica pollinosis is a serious social problem in Japan, affecting almost 20% of the population. In 1992, a genetically male-sterile C. japonica tree whose sterility is determined by a major recessive gene (ms1) was found in Toyama prefecture [13, 14]; this gene is expected play an important role in breeding for reduced pollen dispersal. Since the discovery of this male-sterile individual, considerable effort has been expended on characterizing male sterility in C. japonica, identifying male-sterile and plus-trees, creating artificial crosses between male-sterile and plus-trees, and propagating male-sterile trees . Male-sterile trees are currently identified by direct inspection of the male strobili using a magnifying glass or a microscope. Conversely, plus-trees that are heterozygous for ms1 have been identified by examining segregation data for the progeny arising from artificial crosses; such trees provide important breeding material for seed production while avoiding the problems associated with inbreeding depression. However, these methods are very time-, labour-, and space-intensive; as such, it would be extremely useful to develop a MAS-based method for selecting trees carrying the male-fertile gene.
A composite linkage map for C. japonica was constructed using data for two pedigrees, YI and KO . A total of 438 markers were assigned to 11 large linkage groups and some small or non-integrated linkage groups; the total observed map length was 1372.2 cM, and the average marker interval was 3.0 cM. In general, the most efficient way to study the linkage of a given target gene in a mapping population for which no linkage map is available is to start with a high-density linkage map.
In a recent study on spruces, it was found that the Golden Gate single nucleotide polymorphism (SNP) assay system developed by Illumina greatly facilitates the genetic mapping of species whose genomes have not been extensively studied . To design an SNP genotyping array, it is necessary to identify a large number of SNPs. In the case of C. japonica, a database of 55,543 expressed sequence tags (ESTs) has been constructed from cDNA libraries obtained from seedlings, inner bark, female strobili, male strobili, pollen, leaves, vegetative buds and heartwood; this database is freely-available on the internet (ForestGEN; http://forestgen.ffpri.affrc.go.jp/en/info_cj.html) [18–23]. A number of cDNA-based sequence-tagged site (STS) markers have been also identified using these ESTs [24–26]. A library of C. japonica SNPs suitable for use in a Golden Gate SNP array has been identified , and complements the existing C. japonica EST databases, which will also be useful in identifying candidate genes associated with male gametophyte development and male sterility on the basis of sequence similarity and microarray expression analysis.
This paper reports a study in which a high-density linkage map for C. japonica was constructed and the mapping population was subjected to the GoldenGate genotyping assay. Using information from the high-density linkage map, we mapped the ms1 gene and constructed a partial linkage map around the ms1 locus. This allowed us to identify SNP markers that are tightly linked to the ms1 gene for use in MAS. The paper also includes a discussion of the importance of high-density maps and MAS markers in tree breeding.