Search

Biogenic volatile organic compounds emitted from plants are important constituents of atmospheric chemistry and play a major role in the resistance of plants against various environmental stresses. However, little is known about how abiotic and biotic environments on a geographic scale relate to diversifications of the emission. Here, we present variations of terpenes stored in and emitted from leaves of a single species in a common garden, using genetically differentiated local populations of Japanese cedar, the most dominant and widely distributed tree species in Japan. Furthermore, we determined the composition of fungal communities in 50 locations, based on the presence or absence of 158 fungal species inhabiting the cedar. The results showed that terpenoids, especially those that are emitted, were highly diversified and geographically structured among the 12 populations. The total amount of stored terpenes was negatively affected by warm and less-snow climates. On the other hand, variations in some emitted terpenoid species among the populations were correlated to antagonistic fungal species inhabiting the Japanese cedar. We propose that the diversification of composition and amount of stored and emitted terpenoids in the tree species is not only structured by climate, but also antagonistic fungal communities through biological interactions.

The main purpose of this study was to investigate how a shift in seed sources owing to the implementation of a tree improvement program affected the genetic diversity of plantation forests. We investigated the genetic diversity and pairwise relatedness between individuals in sugi (Cryptomeria japonica D Don) plantations established before (“Pre-B”) and after (“Post-B”) the start of the Japanese tree improvement program. Based on SSR and SNP markers, most genetic diversity indices and the degree of relatedness within plantations were comparable between “Pre-B” and “Post-B”. However, there were large variations in the proportion of closely related pairs within plantations in both groups. Additionally, in “Post-B” plantations, parental contributions greatly diverged among plus-tree clones. The results of this study indicated that the shift in the seed sources, caused by the implementation of a tree improvement program, did not have a significant effect on the genetic diversity of plantations. However, the results also indicated that the degree of relatedness within plantations varied significantly according to how seeds are collected regardless of the type of seed sources. Furthermore, it was also revealed that genetic diversity in “Post-B” plantations could have decreased because of a deviation from equal parental contributions in seed orchards. Our results indicated that effective management of seed orchards is important to secure the genetic diversity of plantation forests.

Cryptomeria japonica is a major forestry tree species in Japan. Male sterility of the species is caused by a recessive gene, which shows dysfunction of pollen development and results in no dispersed pollen. Because the pollen of C. japonica induces pollinosis, breeding of pollen-free C. japonica is desired. In this study, single nucleotide polymorphism (SNP) markers located at 1.78 and 0.58 cM to a male sterility locus (MS1) were identified from an analysis of RNA-Seq and RAD-Seq, respectively. SNPs closely linked to MS1 were first scanned by a method similar to MutMap, where a type of index was calculated to measure the strength of the linkage between a marker sequence and MS1. Linkage analysis of selected SNP markers confirmed a higher efficiency of the current method to construct a partial map around MS1. Allele-specific PCR primer pair for the most closely linked SNP with MS1 was developed as a codominant marker, and visualization of the PCR products on an agarose gel enabled rapid screening of male sterile C. japonica. The allele-specific primers developed in this study would be useful for establishing the selection of male sterile C. japonica.

Pyramiding of male-sterile genes in Cryptomeria japonica is currently being carried out in Niigata prefecture, Japan. This is the first attempt to apply pyramid breeding to forest trees. As the breeding materials for male sterility are limited, special attention must be given to increased genetic relatedness in the process of pyramid breeding to avoid the effects of inbreeding depression as much as possible. In this study, we estimated genetic relatedness based on 246 genome-wide SNP markers for male-sterile individuals in Niigata Prefecture (n = 6) and individuals doubly heterozygous for two male-sterile genes (hereafter referred to as “double-hetero”) produced by marker- assisted selection (n = 124). The pairwise relatedness estimates between male-sterile individuals selected from the same area in Niigata Prefecture were low (−0.01 ± 0.08, mean ± standard deviation), suggesting that there will be almost no negative effects even if the F1 of these male-sterile individuals is used for artificial crossing. On the other hand, the pairwise relatedness between double-hetero individuals in this study was higher than the theoretical relatedness values, as individuals with the slightly higher relatedness were used as parents in artificial crossings. However, there was a large variance in pairwise relatedness for double-hetero individuals. This result suggested that it may be possible to avoid the adverse effects of inbreeding depression by using a pair of double-heteros with lower relatedness for artificial crossing, when we produce a double-homo using the limited breeding materials of male- sterile individuals. It will also be important to continue additional selection of new breeding material for male sterility.

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.

Objective Due to the allergic nature of the pollen of Cryptomeria japonica, the most important Japanese forestry conifer, a pollen-free cultivar is preferred. Mutant trees detected in nature have been used to produce a pollen-free cultivar. In order to reduce the time and cost needed for production and breeding, we aimed to develop simple diagnostic molecular markers for mutant alleles of the causative gene MALE STERILITY 1 (MS1) in C. japonica to rapidly identify pollen-free mutants. Results We developed PCR and LAMP markers to detect mutant alleles and to present experimental options depending on available laboratory equipment. LAMP markers were developed for field stations, where PCR machines are unavailable. The LAMP method only needs heat-blocks or a water bath to perform the isothermal amplification and assay results can be read by the naked eye. Because the causative mutations were deletions, we developed two kinds of PCR markers, amplified length polymorphism (ALP) and allele specific PCR (ASP) markers. These assays can be visualized using capillary or agarose gel electrophoresis.

ObjectiveDue to the allergic nature of the pollen of Cryptomeria japonica, the most important Japanese forestry conifer, a pollen-free cultivar is preferred. Mutant trees detected in nature have been used for the production of a pollen-free cultivar. In order to reduce the time and cost needed for the production and breeding, we aimed to develop simple diagnostic molecular markers for mutant alleles of the causative gene MALE STERILITY 1 (MS1) in C. japonica. The expected function of this gene, its two dysfunctional mutations, and genetic diversity were described recently in a related study.ResultsWe have developed PCR and LAMP markers to detect mutant alleles and to present experimental options depending on available laboratory equipment. At field stations, where PCR machines are not available, LAMP markers were developed. LAMP only needs heat-blocks or a water bath to perform the isothermal amplification and assay results can be easily seen by eye. Because the causative mutations were deletions, two kinds of PCR markers, amplified length polymorphism (ALP) and allele specific PCR (ASP) markers, were developed. These assays can be carried out by capillary or agarose gel electrophoresis.

Identifying causative genes for a target trait in conifer reproduction is challenging for species lacking whole-genome sequences. In this study, we searched for the male-sterility gene (MS1) in Cryptomeria japonica, aiming to promote marker-assisted selection (MAS) of male-sterile C. japonica to reduce the pollinosis caused by pollen dispersal from artificial C. japonica forests in Japan. We searched for mRNA sequences expressed in male strobili and found the gene CJt020762, coding for a lipid transfer protein containing a 4-bp deletion specific to male-sterile individuals. We also found a 30-bp deletion by sequencing the entire gene of another individual with the ms1. All nine breeding materials with the allele ms1 had either a 4-bp or 30-bp deletion in gene CJt020762, both of which are expected to result in faulty gene transcription and function. Furthermore, the 30-bp deletion was detected from three of five individuals in the Ishinomaki natural forest. From our findings, CJt020762 was considered to be the causative gene of MS1. Thus, by performing MAS using two deletion mutations as a DNA marker, it will be possible to find novel breeding materials of C. japonica with the allele ms1 adapted to the unique environment of each region of the Japanese archipelago.

Recently, there has been a continuous increase in the concentration of tropospheric ozone in urban forests in Japan. Since monoterpenes are precursors to ozone, we need to evaluate the effects of ozone exposure on all tree species that are considered to be sources of monoterpenes. Cryptomeria japonica, which is the most widely planted afforestation tree, is classified into three different gene pools. However, the interclone variation for seasonal changes in the ozone exposure effect has not been evaluated. Thus, free-air ozone enhancement experiments were conducted using three representative clones of C. japonica in the summer and winter. After exposure to twice the ozone concentration in the ambient atmosphere, the effect on the monoterpene emission rate was found to be considerably different among the clones and for the different seasons. The monoterpene emission rate after ozone exposure increased in winter and summer in the native clones (Donden and Yakushima) in the snow area and heavy rain area, respectively. Since monoterpenes are antibacterial substances, each clone adapted sensitivity to stress during each season upon considerable damage. These results suggest that not only differences between tree species but also differences between clones are important for evaluating seasonal variation characteristics after ozone exposure.

Genomic regions which affected tree growth and wood property traits were investigated in the major plantation tree of Japan, Cryptomeria japonica, in three replicated common garden experiments planted in contrasting environments in Kyushu and Honshu, Japan. Phenotypic traits measured were stem diameter at breast height, tree height, wood strength (Young’s modulus), heartwood density, sapwood density, heartwood moisture content, and sapwood moisture content. Quantitative trait locus (QTL) analysis identified an average of 53 QTLs across the three environments. There were two QTLs which affected the same traits across all three environments. These stable QTLs were identified as being associated with sapwood density and Young’s modulus and explained 3.5–11.3% and 2.1–18.7% of the total genotypic variation, respectively. In contrast, the majority of QTLs detected were unique to only one environment, a finding which is consistent with QTL mapping studies of other forest trees, indicating a substantial contribution of environmental effects on the mapping progenies. Nonetheless, the two stable QTLs identified in this study could be important genomic regions to target for further research aimed at maximizing breeding efficiency and wood quality of C. japonica across wide environmental gradients.

Research highlights: Our study is the first to explore the genetic composition of ancient Cryptomeria trees across a distribution range in China. Background and objectives: Cryptomeria japonica var. sinensis is a native forest species of China, it is widely planted in the south of the country to create forests and for wood production. Unlike Cryptomeria in Japan, genetic Chinese Cryptomeria has seldom been studied, although there is ample evidence of its great ecological and economic value. Materials and methods: Because of overcutting, natural populations are rare in the wild. In this study, we investigated seven ancient tree populations to explore the genetic composition of Chinese Cryptomeria through ddRAD-seq technology. Results: The results reveal a lower genetic variation but higher genetic differentiation (Ho = 0.143, FST = 0.1204) than Japanese Cryptomeria (Ho = 0.245, FST = 0.0455). The 86% within-population variation is based on an analysis of molecular variance (AMOVA). Significant excess heterozygosity was detected in three populations and some outlier loci were found, these were considered to be the consequence of selection or chance. Structure analysis and dendrogram construction divided the seven ancient tree populations into four groups corresponding to the geographical provinces in which the populations are located, but there was no obvious correlation between genetic distance and geographic distance. A demographic history analysis conducted by a Stairway Plot showed that the effective population size of Chinese Cryptomeria had experienced a continuing decline from the mid-Pleistocene to the present. Our findings suggest that the strong genetic drift caused by climate fluctuation and intense anthropogenic disturbance together contributed to the current low diversity and structure. Considering the species&rsquo, unfavorable conservation status, strategies are urgently required to preserve the remaining genetic resources.

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.

Local adaptation of plant species is a central issue for survival during global climate change, especially for long-lived forest trees, with their lengthy regeneration time and spatially limited gene flow. Identification of loci and/or genomic regions associated with local adaptation is necessary for knowledge of both evolution and molecular breeding for climate change. Cryptomeria japonica is an important species for forestry in Japan; it has a broad natural distribution and can survive in a range of different environments. The genetic structure of 14 natural populations of this species was investigated using 3930 SNP markers. Populations on the Pacific Ocean side of Japan are clearly different from those on the Japan Sea side, as discussed in previous studies. Structure analysis and population network trees show that peripheral populations, including the most northerly and southerly ones, have unique features. We found that the genetic differentiation coefficient is low, FST = 0.05, although it must account for the presence of important genes associated with adaptation to specific environments. In total, 208 outlier loci were detected, of which 43 were associated with environmental variables. Four clumped regions of outlier loci were detected in the genome by linkage analysis. Linkage disequilibrium (LD) was quite high in these clumps of outlier loci, which were found in linkage groups (LGs) 2, 7, 10, and 11, especially between populations of two varieties, and when interchromosomal LD was also detected. The LG7 region is characteristic of the Yakushima population, which is a large, isolated, peripheral population occupying a specific environment resulting from isolation combined with volcanic activity in the region. The detected LD may provide strong evidence for selection between varieties.

Population structure and genome-wide linkage disequilibrium (LD) were investigated in a Cryptomeria japonica core collection consisting of 510 individuals and providing comprehensive coverage of the natural distribution range of the species, using 4,896 SNP markers. Both Bayesian clustering and principal coordinate analyses clearly subdivided the sample into two distinct groups corresponding to the Japan Sea side and the Pacific Ocean side of Japan. The clustering analysis also detected two subgroups located in Northern Tohoku and Yakushima Island, as was also the case in the natural population. This finding is in accord with the ecogeographical origin of the cultivars; however, the spatial distribution of these clusters was slightly different between the natural population and the core collection, especially in the Chugoku district, an effect which might have resulted from historical human activity. The genetic diversity of the core collection is comparable to, or a little higher than, that of natural populations, indicating that the individuals in the core collection have not suffered from diversity losses caused by a domestication bottleneck. LD analysis with mapped markers indicated that the LD decays rapidly, within approximately 5 cM, in both the core collection and natural populations. The results show that the population described here is a valuable resource for both basic and applied research on C. japonica.

Clonal reproduction is a crucial factor shaping the demography and genetic structure of many plant populations. However, we still do not understand the extent to which environmental and genetic factors influence clonal reproduction. Natural forests of Cryptomeria japonica are distributed under a range of environmental conditions in the Japanese Archipelago. In this study, the spatial genetic structures of 13 natural old-growth C. japonica forest plots of about 0.25 ha were investigated using eight nuclear microsatellite markers. Clonal reproduction was detected in 10 plots, the clonal frequency in each plot varied. Patterns of spatial genetic structure at the ramet level showed significant positive autocorrelation over short distances, which may reflect clonal reproduction and the limited extent to which seedlings establish in gaps. The clonality of individual genets was influenced by the Q value of cluster 1 of the results of STRUCTURE analysis as the genetic factor, snow depth as the environmental factor, the average basal area (BA) and the interaction between Q value and snow depth. This result indicated that clonal genets had low Q values for cluster 1 and small BA, especially in regions experiencing heavy snow. On the other hand, the average BA had a negative effect for a number of ramets, however the parameter estimate was small. Our results suggest that clonal reproduction may have been the most important of the strategies exploited in order to establish in harsh environments like frequent disturbance conditions such as heavy snow region in this species.

Long-distance dispersal (LDD) of seeds has a critical impact on species survival in patchy landscapes. However, relative to pollen dispersal, empirical data on how seed LDD affects genetic diversity in fragmented populations have been poorly reported. Thus, we attempted to indirectly evaluate the influence of seed LDD by estimating maternal and paternal inbreeding in the seed rain of fragmented 8 Pinus densiflora populations. In total, the sample size was 458 seeds and 306 adult trees. Inbreeding was estimated by common parentage analysis to evaluate gene flow within populations and by sibship reconstruction analysis to estimate gene flow within and among populations. In the parentage analysis, the observed probability that sampled seeds had the same parents within populations was significantly larger than the expected probability in many populations. This result suggested that gene dispersal was limited to within populations. In the sibship reconstruction, many donors both within and among populations appeared to contribute to sampled seeds. Significant differences in sibling ratios were not detected between paternity and maternity. These results suggested that seed-mediated gene flow and pollen-mediated gene flow from outside population contributed some extent to high genetic diversity of the seed rain (H E > 0.854). We emphasize that pine seeds may have excellent potential for gene exchange within and among populations.

Cryptomeria japonica pollinosis is one of the most serious allergic diseases in Japan; this is a social problem because C. japonica is the most important Japanese forestry species. In order to reduce the amount of pollen dispersed, breeding programs using trees with male-sterile genes have been implemented. High-density linkage maps with stable ordering of markers facilitate the localization of male-sterile genes and the construction of partial linkage maps around them in order to develop markers for use in marker-assisted selection. In this study, a high-density linkage map for C. japonica with 2560 markers was constructed. The observed map length was 1266.2 cM and the mean distance between adjacent markers was 0.49 cM. Using information from this high-density map, we newly located two male-sterile genes (ms3 and ms4) on the first and fourth linkage groups, respectively, and constructed partial linkage maps around these loci. We also constructed new partial linkage maps around the ms1 and ms2 loci using additional SNP markers. The closest markers to the ms1, ms2, ms3, and ms4 male-sterile loci were estSNP04188 (1.8 cM), estSNP00695 (7.0 cM), gSNP05415 (3.1 cM), and estSNP01408 (7.0 cM) respectively. These results allowed us to develop SNP markers tightly linked to the male sterile genes for use in MAS; this will accelerate the future isolation of these genes by map-based cloning approaches.

Local adaptation is important in evolutionary processes and speciation. We used multiple tests to identify several candidate genes that may be involved in local adaptation from 1026 loci in 14 natural populations of Cryptomeria japonica, the most economically important forestry tree in Japan. We also studied the relationships between genotypes and environmental variables to obtain information on the selective pressures acting on individual populations. Outlier loci were mapped onto a linkage map, and the positions of loci associated with specific environmental variables are considered. The outlier loci were not randomly distributed on the linkage map; linkage group 11 was identified as a genomic island of divergence. Three loci in this region were also associated with environmental variables such as mean annual temperature, daily maximum temperature, maximum snow depth, and so on. Outlier loci identified with high significance levels will be essential for conservation purposes and for future work on molecular breeding.

† Background and Aims Distribution shifts and natural selection during past climatic changes are important factors in determining the genetic structure of forest species. In particular, climatic fluctuations during the Quaternary appear to have caused changes in the distribution ranges of plants, and thus strongly affected their genetic structure. This study was undertaken to identify the responses of the conifer Cryptomeria japonica, endemic to the Japanese Archipelago, to past climatic changes using a combination of phylogeography and species distribution modelling (SDM) methods. Specifically, this study focused on the locations of refugia during the last glacial maximum (LGM). † Methods Genetic diversity and structure were examined using 20 microsatellite markers in 37 populations of C. japonica. The locations of glacial refugia were assessed using STRUCTURE analysis, and potential habitats under current and past climate conditions were predicted using SDM. The process of genetic divergence was also examined using the approximate Bayesian computation procedure (ABC) in DIY ABC to test the divergence time between the gene pools detected by the STRUCTURE analysis. † Key Results STRUCTURE analysis identified four gene pools: northern Tohoku district; from Chubu to Chugoku district; from Tohoku to Shikoku district on the Pacific Ocean side of the Archipelago; and Yakushima Island. DIY ABC analysis indicated that the four gene pools diverged at the same time before the LGM. SDM also indicated potential northern cryptic refugia. † Conclusions The combined evidence from microsatellites and SDM clearly indicates that climatic changes have shaped the genetic structure of C. japonica. The gene pool detected in northern Tohoku district is likely to have been established by cryptic northern refugia on the coast of the Japan Sea to the west of the Archipelago. The gene pool in Yakushima Island can probably be explained simply by long-term isolation from the other gene pools since the LGM. These results are supported by those of SDM and the predicted divergence time determined using ABC analysis.

Background High-density linkage maps facilitate the mapping of target genes and the construction of partial linkage maps around target loci to develop markers for marker-assisted selection (MAS). MAS is quite challenging in conifers because of their large, complex, and poorly-characterized genomes. Our goal was to construct a high-density linkage map to facilitate the identification of markers that are tightly linked to a major recessive male-sterile gene (ms1) for MAS in C. japonica, a species that is important in Japanese afforestation but which causes serious social pollinosis problems. Results We constructed a high-density saturated genetic linkage map for C. japonica using expressed sequence-derived co-dominant single nucleotide polymorphism (SNP) markers, most of which were genotyped using the GoldenGate genotyping assay. A total of 1261 markers were assigned to 11 linkage groups with an observed map length of 1405.2 cM and a mean distance between two adjacent markers of 1.1 cM; the number of linkage groups matched the basic chromosome number in C. japonica. Using this map, we located ms1 on the 9th linkage group and constructed a partial linkage map around the ms1 locus. This enabled us to identify a marker (hrmSNP970_sf) that is closely linked to the ms1 gene, being separated from it by only 0.5 cM. Conclusions Using the high-density map, we located the ms1 gene on the 9th linkage group and constructed a partial linkage map around the ms1 locus. The map distance between the ms1 gene and the tightly linked marker was only 0.5 cM. The identification of markers that are tightly linked to the ms1 gene will facilitate the early selection of male-sterile trees, which should expedite C. japonica breeding programs aimed at alleviating pollinosis problems without harming productivity.

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.

Background Microsatellites or simple sequence repeats (SSRs) in expressed sequence tags (ESTs) are useful resources for genome analysis because of their abundance, functionality and polymorphism. The advent of commercial second generation sequencing machines has lead to new strategies for developing EST-SSR markers, necessitating the development of bioinformatic framework that can keep pace with the increasing quality and quantity of sequence data produced. We describe an open scheme for analyzing ESTs and developing EST-SSR markers from reads collected by Sanger sequencing and pyrosequencing of sugi (Cryptomeria japonica). Results We collected 141,097 sequence reads by Sanger sequencing and 1,333,444 by pyrosequencing. After trimming contaminant and low quality sequences, 118,319 Sanger and 1,201,150 pyrosequencing reads were passed to the MIRA assembler, generating 81,284 contigs that were analysed for SSRs. 4,059 SSRs were found in 3,694 (4.54%) contigs, giving an SSR frequency lower than that in seven other plant species with gene indices (5.4–21.9%). The average GC content of the SSR-containing contigs was 41.55%, compared to 40.23% for all contigs. Tri-SSRs were the most common SSRs; the most common motif was AT, which was found in 655 (46.3%) di-SSRs, followed by the AAG motif, found in 342 (25.9%) tri-SSRs. Most (72.8%) tri-SSRs were in coding regions, but 55.6% of the di-SSRs were in non-coding regions; the AT motif was most abundant in 3′ untranslated regions. Gene ontology (GO) annotations showed that six GO terms were significantly overrepresented within SSR-containing contigs. Forty–four EST-SSR markers were developed from 192 primer pairs using two pipelines: read2Marker and the newly-developed CMiB, which combines several open tools. Markers resulting from both pipelines showed no differences in PCR success rate and polymorphisms, but PCR success and polymorphism were significantly affected by the expected PCR product size and number of SSR repeats, respectively. EST-SSR markers exhibited less polymorphism than genomic SSRs. Conclusions We have created a new open pipeline for developing EST-SSR markers and applied it in a comprehensive analysis of EST-SSRs and EST-SSR markers in C. japonica. The results will be useful in genomic analyses of conifers and other non-model species.