Genetic diversity and the genetic structure of natural populations of Chamaecyparis obtusa: implications for management and conservation.

We investigated 25 natural populations of Chamaecyparis obtusa using 51 cleaved amplified polymorphic sequence (CAPS) markers, which were developed using information on sequence-tagged sites (STS) in Cryptomeria japonica. Most CAPS markers have codominant expression patterns, and are suitable for population studies because of their robustness and convenience. We estimated various genetic diversity parameters, including average heterozygosity (H(e)) and allelic richness and found that the more peripheral populations tended to have lower genetic diversity than central populations, in agreement with a previous theoretical study. The overall genetic differentiation between populations was low, but statistically significant (G(ST)=0.039), and similar to the level reported in a previous allozyme study. We attempted to detect non-neutral loci associated with local adaptation to clarify the relationship between the fixation index (F(ST)) and H(e) values for each locus and found seven candidates non-neutral loci. Phylogenetic tree analysis of the populations and Bayesian clustering analysis revealed a pattern of gradually increasing isolation of populations with increasing geographical distance. Three populations had a high degree of linkage disequilibrium, which we attribute to severe bottlenecks due to human disturbance or competition with other species during their migration from refugia after the most recent glaciation. We concluded that the small populations in western Japan and in Kanto district are more important, from a conservation perspective, than the populations in central Japan, due to their genetic divergence, relatively small sizes and restricted areas.

Development and characterization of microsatellite markers for Cryptomeria japonica D.Don.

Thirty four microsatellite markers for Cryptomeria japonica D. Don were developed by searching three types of library: a database of C. japonica cDNA sequences, a standard non-enriched genomic DNA library and a microsatellite-enriched library using magnetic particles. The enrichment of microsatellite sequences using magnetic particles is very efficient compared to the other two methods both in terms of the numbers of markers generated, and in the polymorphism they detect. The microsatellites developed from the genomic DNA library generally have longer repeat sequences and are more polymorphic than those from cDNA. All the developed microsatellite markers in this study showed polymorphism among 28 plus trees selected from locations scattered throughout Japan. The mean number of alleles per locus (MNA) detected in the 28 plus trees ranged from 2 to 21 with an average of 7.5. The Polymorphism Information Content (PIC) ranged from 0.160 to 0.936 with an average of 0.666. Co-dominant segregation of alleles in a three-generation pedigree of C. japonica was demonstrated at 34 microsatellite loci, and the segregation was not distorted from Mendelian expectation for all loci. In 12 out of 34 loci, a null allele was detected. Key relationships between polymorphic parameters, such as MNA and PIC, and the characteristics of microsatellite sequences, such as the longest repeat number, total repeat number and total number of nucleotides, were investigated using rank correlation coefficients, Kendall's tau. A positive correlation was found between repeat lengths and polymorphisms. The markers provide sufficient resolution for investigating gene flow within forests and seed orchards, and for genome mapping.

Expression analysis of ESts derived from the inner bark of Cryptomeria japonica.

To assist genetic research into Cryptomeria japonica, which is one of the most important forest tree species in Japan, expressed sequence tag (EST) analysis was carried out. The cDNA clones were isolated from a library derived from inner bark tissues. Partial sequences were obtained from 2231 clones, representing 1398 unique transcripts. Putative functions were assigned to 1583 clones, which represented 882 unique transcripts, by a Blast algorithm. Homology analysis suggested that ESTs related to cell wall formation represented about 3% of the clones. Transcripts of plant stress response genes were also abundant in the inner bark library, especially genes involved in wounding and drought responses. This indicates that the stress response systems of this tree species are similar to those of other plants, and that these systems are highly conserved among plant species. The remaining 648 clones, which represented 516 unique transcripts, did not show any significant homology to known sequences in the databases searched: these are expected to represent genes specific to Cryptomeria and, possibly, to related species.