Yuzuru Mukai

Sequence-tagged-sites (STSs) of cDNA clones in Cryptomeria japonica and their evaluation as molecular markers in conifers

Abstract We have generated 66 sequence-tagged-site (STS) markers from cDNA clones of Cryptomeria japonica, and 60% of them have already been mapped into C. japonica linkage groups. All of the STS markers showed a single fragment following polymerase chain reaction (PCR) amplification. We investigated by polymorphism of these STS markers in a mapped F2 population and 15 plus trees by means of a restriction endonuclease analysis. Polymorphism levels were 10.6% and 22.7% in the F2 population and the 15 plus trees, respectively. PCR amplification levels of the 66 STS markers in 14 conifer species varied depending on their genetic relationship with C. japonica. Taxodium, which is closely related to C. japonica, had the most amplifications (31.82%), followed by Sequoiadendron giganteum, which is of the same family. The average proportion of PCR amplifications in each family gradually declined in the following order: from Taxodiaceae to Cuppresaceae, Sciadopityaceae, Pinaceae, and Taxaceae. These results are in general agreement with a molecular phylogenetic relationship based on chloroplast DNA. The 66 STS markers will be useful as on anchor point for genome mapping and population genetics, and some of them will also be useful when studying other conifers.

A linkage map for sugi (Cryptomeria japonica) based on RFLP, RAPD, and isozyme loci

A linkage map for sugi was constructed on the basis of restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), and isozyme loci using a three-generation pedigree prepared for genetic analysis of heartwood color. A total of 128 RFLP (123 cDNA and 5 genomic probes), 33 RAPD, 2 isozyme, and 1 morphological (dwarf) loci segregated in 73 progeny. Of the 164 segregating loci, 145 loci were distributed in 20 linkage groups. Of these loci, 91 with confirmed map positions were assigned to 13 linkage groups, covering a total of 887.3 cM. A clustering of markers with distorted segregation was observed in 6 linkage groups. In the four clusters, distortions with a reduction in the number of homozygotes from one parent only were found.

Cleaved amplified polymorphic sequence markers in sugi, Cryptomeria japonica D. Don, and their locations on a linkage map

Abstract  Sugi, Cryptomeria japonica D. Don, is one of the most important forestry species in Japan. We here report the development of 217 CAPS markers derived from sugi cDNA libraries. More than half of a set of STS markers produced could be converted into CAPS markers using restriction endonuclease analysis. Of the 217 markers, 71 showed different patterns of polymorphism when they were digested with a range of endonucleases and, in total, 347 polymorphisms were found in the various combinations of STSs and endonucleases. When the polymorphisms gave co-dominant patterns in a screening program, the polymorphic information content (PIC) used to evaluate the value of the polymorphisms was relatively high (0.33, on average) compared to the information yielded by commonly used markers, like isozymes. The results of a segregation analysis suggest that approximately 80% of the CAPS markers developed here will show co-dominant inheritance. From logistic regression analysis, the polymorphisms were found to be associated more strongly with intron than with exon regions. Sixty two markers were subsequently mapped on the previously reported linkage map, 15 of which showed abnormal segregation, presumably caused by linkage with lethal factors.

Comparative analysis of expressed sequence tags of conifers and angiosperms reveals sequences specifically conserved in conifers

To identify and characterize lineage-specific genes of conifers, two sets of ESTs (with 12791 and 5902 ESTs, representing 5373 and 3018 gene transcripts, respectively) were generated from the Cupressaceae species Cryptomeria japonica and Chamaecyparis obtusa. These transcripts were compared with non-redundant sets of genes generated from Pinaceae species, other gymnosperms and angiosperms. About 6% of tentative unique genes (Unigenes) of C. japonica and C. obtusa had homologs in other conifers but not angiosperms, and about 70% had apparent homologs in angiosperms. The calculated GC contents of orthologous genes showed that GC contents of coniferous genes are likely to be lower than those of angiosperms. Comparisons of the numbers of homologous genes in each species suggest that copy numbers of genes may be correlated between diverse seed plants. This correlation suggests that the multiplicity of such genes may have arisen before the divergence of gymnosperms and angiosperms.

Genetic structure of island populations of Prunus lannesiana var. speciosa revealed by chloroplast DNA, AFLP and nuclear SSR loci analyses

The wild flowering cherry Prunuslannesiana var. speciosa is highly geographically restricted, being confined to the Izu Islands and neighboring peninsulas in Japan. In an attempt to elucidate how populations of this species have established we investigated the genetic diversity and differentiation in seven populations (sampling 408 individuals in total), using three kinds of genetic markers: chloroplast DNA (cpDNA), amplified fragment length polymorphisms (AFLPs), and 11 nuclear SSR polymorphic loci. Eight haplotypes were identified based on the cpDNA sequence variations, 64 polymorphic fragments were scored for the AFLP markers, and a total of 154 alleles were detected at the 11 nuclear SSR loci. Analysis of molecular variance showed that among-population variation accounted for 16.55, 15.04 and 7.45% of the total detected variation at the cpDNA, AFLPs, and SSR loci, respectively. Thus, variation within populations accounted for most of the genetic variance for all types of markers, although the genetic differentiation among populations was also highly significant. For cpDNA variation, no clear structure was found among the populations, except that of the most distant island, although an “isolation by distance” pattern was found for each marker. Both neighbor-joining trees and structure analysis indicate that the genetic relationships between populations reflect geological variations between the peninsula and the islands and among the islands. Furthermore, hybridization with related species may have affected the genetic structure, and some genetic introgression is likely to have occurred.

Isolation and Characterization of cDNAs That Encode Homologs of a Pathogenesis-related Protein Allergen from Cryptomeria japonica

Many plant pathogenesis-related (PR) proteins are allergenic. We isolated three cDNAs, Cry j 3.1, Cry j 3.2, and Cry j 3.3, that encoded homologs of Jun a 3, a PR protein allergen in Juniperus ashei, from a cDNA library derived from the pollen of Cryptomeria japonica. The predicted amino acid sequences encoded by the three cDNAs were more than 85% identical to each other and about 57% identical to the sequence of Jun a 3. The Cry j 3 genes seemed to form a small multigene family in the genome of C. japonica. Expression of Cry j 3 was strong in roots and in female and male strobili; expression was weaker in cotyledons, leaves, stems, and pollen grains.

Agrobacterium tumefaciens-mediated transformation of Japanese white birch (Betula platyphylla var. japonica)

Abstract Genetically transformed Japanese white birch ( Betula platyphylla var. japonica ) plants were regenerated after co-cultivation of leaf discs with Agrobacterium tumefaciens strain LBA4404 that harbored a binary vector (pBI121) which included genes for β -glucuronidase (GUS) and neomycin phosphotransferase. The efficiency of Agrobacterium tumefaciens -mediated transformation of Japanese white birch was markedly increased by the inclusion of acetosyringone in the co-cultivation medium. Successful transformation was confirmed by the ability of leaf discs to produce calli in the presence of kanamycin, by histochemical and fluorometric assays of GUS activity in plant tissues, and by Southern blot analysis. With this transformation system, about 4 months were required for regeneration of the transgenic birch plants from leaf discs. The frequency of transformation was approximately 18%.

Seasonal changes in photosynthesis and nitrogen allocation in leaves of different ages in evergreen understory shrub Daphniphyllum humile

Seasonal changes in photosynthetic capacity, leaf nitrogen (N) content, leaf chlorophyll (Chl) content and leaf N allocation patterns in leaves of different ages in the evergreen understory shrub, Daphniphyllumhumile Maxim, growing at a forest border and an understory site were studied. In current-year leaves at the understory site, the N and Rubisco contents increased from spring to autumn although their light-saturated photosynthetic rate at 22°C (Pmax22) remained stable, indicating that their mesophyll conductance rates declined as they completed their development and/or that they invested increasing amounts of their resources in photosynthetic enzymes during this period. In contrast, seasonal changes in Pmax22 in current-year leaves at the forest border site were correlated with changes in Rubisco content. In 1-year old leaves at the understory site, Pmax22 and contents of Chl, leaf N, and Rubisco remained stable from spring to autumn, while these parameters decreased in 1-year-old forest border leaves, indicating that N may have been remobilized from shaded 1-year-old leaves to sunlit current-year leaves. When leaves senesced at the forest border site the Rubisco content decreased more rapidly than that of light-harvesting proteins such as LHCII, suggesting that N remobilization from Rubisco may be more efficient, possibly because Rubisco has greater N costs and is soluble, whereas the light-harvesting proteins are membrane components.

Genetic structure of Cerasus jamasakura, a Japanese flowering cherry, revealed by nuclear SSRs : implications for conservation

The genetic resources of a particular species of flowering cherry, Cerasus jamasakura, have high conservation priority because of its cultural, ecological and economic value in Japan. Therefore, the genetic structures of 12 natural populations of C. jamasakura were assessed using ten nuclear SSR loci. The population differentiation was relatively low (FST, 0.043), reflecting long-distance dispersal of seeds by animals and historical human activities. However, a neighbor-joining tree derived from the acquired data, spatial analysis of molecular variance and STRUCTURE analysis revealed that the populations could be divided into two groups: one located on Kyusyu Island and one on Honshu Island. Genetic diversity parameters such as allelic richness and gene diversity were significantly lower in the Kyushu group than the Honshu group. Furthermore, STRUCTURE analysis revealed that the two lineages were admixed in the western part of Honshu Island. Thus, although the phylogeographical structure of the species and hybridization dynamics among related species need to be evaluated in detail using several marker systems, the Kyusyu Island and Honshu Island populations should be considered as different conservation units, and the islands should be regarded as distinct seed transfer zones for C. jamasakura, especially when rapid assessments are required.

Origins of Japanese flowering cherry (Prunus subgenus Cerasus) cultivars revealed using nuclear SSR markers

Japanese flowering cherry (Prunus subgenus Cerasus) cultivars, which are characterized by beautiful flowers, have been developed through hybridization among wild Prunus taxa. The long history of cultivation has caused significant confusion over the origins of these cultivars. We conducted molecular analysis using nuclear simple sequence repeat (SSR) polymorphisms to trace cultivar origins. Bayesian clustering based on the STRUCTURE analysis using SSR genotypes revealed that many cultivars originated from hybridization between two or more wild species. This suggests that morphological variations among flowering cherry cultivars probably arose through a complex sequence of hybridizations. Our findings generally supported estimates of the origins of cultivars based on morphological study, although there were some exceptions.