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). 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. 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.

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. 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 9^th 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. Using the high-density map, we located the ms1 gene on the 9^th 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.

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, F_ST = 0.1204) than Japanese Cryptomeria (Ho = 0.245, F_ST = 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’ unfavorable conservation status, strategies are urgently required to preserve the remaining genetic resources.

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.

Research Highlights: We detected the longitudinal gradients of genetic diversity parameters, such as the number of alleles, effective number of alleles, heterozygosity, and inbreeding coefficient, and found that these might be attributable to climatic conditions, such as temperature and snow depth. Background and Objectives: Genetic diversity among local populations of a plant species at its distributional margin has long been of interest in ecological genetics. Populations at the distribution center grow well in favorable conditions, but those at the range margins are exposed to unfavorable environments, and the environmental conditions at establishment sites might reflect the genetic diversity of local populations. This is known as the central-marginal hypothesis in which marginal populations show lower genetic variation and higher differentiation than in central populations. In addition, genetic variation in a local population is influenced by phylogenetic constraints and the population history of selection under environmental constraints. In this study, we investigated this hypothesis in relation to Abies sachalinensis, a major conifer species in Hokkaido. Materials and Methods: A total of 1189 trees from 25 natural populations were analyzed using 19 EST-SSR loci. Results: The eastern populations, namely, those in the species distribution center, showed greater genetic diversity than did the western peripheral populations. Another important finding is that the southwestern marginal populations were genetically differentiated from the other populations. Conclusions: These differences might be due to genetic drift in the small and isolated populations at the range margin. Therefore, our results indicated that the central-marginal hypothesis held true for the southernmost A. sachalinensis populations in Hokkaido.

Summary: Introgression from one species in a specific environment to another may facilitate colonization of the environment by the recipient species. However, such environment‐dependent introgression has been clarified in limited plant taxa. In northern Japan, there are two interfertile oak species: Quercus dentata (Qd) in coastal areas and Q. mongolica var. crispula (Qc) in inland areas. However, at higher latitudes where Qd is rare, a coastal Qc ecotype with Qd‐like traits is distributed in the coastal areas. We distinguished inland Qc, coastal Qc, and coastal Qd populations based on genome‐wide genotypes and multitrait phenotypes and verified introgression from coastal Qd to coastal Qc using reduced library sequencing. Genotypes and phenotypes differed among the populations, and coastal Qc was intermediate between inland Qc and coastal Qd. The ABBA–BABA test showed introgression from coastal Qd to coastal Qc. In coastal Qc, we found various stages of introgression after the first generation of backcross but detected no genomic regions where introgression was enhanced. Overall, we show evidence for introgression from a coastal species to an ecotype of an inland species, which has colonized the coastal environment. It remains unclear whether introgressed alleles are selected in the coastal environment.