Kihachiro Ohba

Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir (Pseudotsuga menziesii) and sugi (Cryptomeria japonica)

We studied inter-simple sequence repeat (ISSR) polymorphism and inheritance in Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] and sugi (Cryptomeria japonica D. Don) megagametophytes using primers that anneal to simple repeats of various lengths, sequences, and non-repetitive motifs at the 5′ and 3′ ends. Products were visualized on agarose gels with ethidium bromide staining. More than 60% of the 96 primers tested gave interpretable banding patterns in both Douglas-fir and sugi, and the useful primers were in complete agreement among species. Dinucleotide repeat primers were the majority of those tested, and gave all of the useful banding patterns. The 24 best primers were used for segregation studies, yielding a total of 77 loci distributed among two Douglas-fir families and one sugi family. Approximately 90% of the 24 primers showed polymorphism within at least one of the three families. The average number of variable loci per primer was 1.6. Primers based on (AG)n repeats gave the largest number of polymorphic loci; 16 primer-family combinations yielded 24 segregating loci. However, primer based on (GT)n repeats gave the most loci per primer studied (mean of 2.0). All markers displayed apparent dominance (band presence vs absence), and all but three segregation ratios (4%) fit Mendelian expectations: Because they employ longer primers than do RAPDs, have a high degree of polymorphism, conform well to Mendelian expectations, and do not require use of acrylamide gels for analysis, ISSRs may be useful markers for PCR-based genome maps and population studies of conifers.

Molecular phytogeny of conifers using RFLP analysis of PCR-amplified specific chloroplast genes.

We investigated the molecular phylogeny of conifers using restriction endonuclease fragment length polymorphism of six polymerase chain reaction-amplified chloroplast genes — frxC, rbcL, psbA, psbD, trnK, and 16S. We detected 227 total site changes among species, representing 23, 26, 38, 48, 67, and 25 site changes in frxC, psbA, psbD, rbcL, trnK and 16S, respectively. The mean nucleotide substitution was 10.75% (SD 0.573) among species in five families. Forty maximally parsimonious trees were obtained using the Wagner parsimony method, and a 50% majority-rule consensus tree was obtained from them. Data analysis produced similar basic patterns when both the Wagner parsimony and the neighbor-joining methods were applied, and the main lineages were clearly separated. Taxaceae and Cephalotaxaceae species were used as the out-groups when applying Wagner parsimony methods. With the Wagner method, the consistency index was 0.510, the retention index was 0.879, and tree length was 435 steps. Our results indicated that Cupressaceae and Taxodiaceae are closely related families and that Sciadopitys verticillata is the basal lineage of Cupressaceae and Taxodiaceae. The neighbor-joining tree is similar to the 50% majority-rule consensus of the 40 Wagner parsimony trees except for the position of Keteleeria daversifolia, the Picea and Cedrus group, and the divergence within Cupressaceae.

GENETIC DIVERSITY IN FAGUS CRENATA (JAPANESE BEECH) : INFLUENCE OF THE DISTRIBUTIONAL SHIFT DURING THE LATE-QUATERNARY

Genetic diversity at 11 loci encoding nine enzymes was studied in 23 populations of Japanese beech Fagus crenata Blume distributed throughout the range of the species. Levels of genetic diversity were high for both within species (expected mean heterozygosity: 0.194) and within populations (expected mean heterozygosity: 0.187), whereas the level of genetic diversity among populations was low (GST = 0.038), as observed in various long-lived, woody plants. Despite the low differentiation among populations, geographical patterning of the variation was observed. Populations in south-western Japan tended to have greater within-population variation and to be more highly differentiated when compared with those in north-eastern Japan. In addition, allele frequencies observed at eight loci were significantly related to latitudinal and/or longitudinal gradients and showed clinal variation across the range of the species. Principal components analysis revealed that the populations tended to cluster according to their geographical locations. The nonrandom patterns of variation were probably shaped by relatively recent historical events such as late-Quaternary migration and founding events.

Germplasm conservation of the tropical forest trees,Cedrela odorata L.,Guazuma crinita Mart., andJacaranda mimosaefolia D. Don., by shoot tip encapsulation in calcium-alginate and storage at 12–25°C

Germplasm conservation of the tropical forest trees,Cedrela odorata L.,Guazuma crinita Mart., andJacaranda mimosaefolia D. Don., at above-freezing temperatures following alginate-bead encapsulation was attempted. Shoot tips excised from in vitro plantlets were encapsulated in calcium-alginate beads and stored on different substrates at 12, 20, and 25 °C. Percent viability when encapsulated shoot tips were stored on substrate containing only water solidified with 1% (wt/vol) agar was 80% after 12 months at 12°C forC. odorata, 90% after 12 months at 25°C forG. crinita, and 70% after 6 months at 20°C forJ. mimosaefolia.

Genetic structure within a Japanese stone pine (Pinus pumila regel) population on Mt. Aino-dake in central Honshu, Japan

In previous investigations, natural layering of Japanese stone pine (Pinus pumila) was suggested by the occurrence of adventitious roots. However, there is no genetic evidence so far that this species actually produces offspring by natural layering. We, therefore, investigated clonal structure and spatial genetic structure within a 38×18 m plot on Mt. Aino-dake, using allozyme, random amplified polymorphic DNA (RAPD), and inter-simple sequence repeat (ISSR) analyses. We found 24 genets, with stems found to be genetically identical in multiple tests, which extended later-ally against the direction of the slope, indicating that there were clonal structures originating from elongation of ramified stems and subsequent natural layering. The results suggest, however, that less than one third of the 200 stems analyzed from this site were clonaly propagated. We also analyzed spatial genetic structure by spatial autocorrelation. Many of the spatial autocorrelation coefficients were significantly positive in short distance classes. We concluded that the species has genetic structures which largely originate from clonal propagation and avian seed dispersal.

Geographical cline of chloroplast DNA variation in Abies mariesii.

Where its populations are isolated in higher mountain regions, Abies mariesii is one of the more important conifers of Japans alpine forest zone. In this study we tried to clarify the genetic variation of chloroplast DNA (cpDNA) in A. mariesii. Cones and fresh needles were collected from seven mountain regions. Total DNAs were extracted from individual seedlings, and these were digested by 15 restriction endonucleases. Southern hybridization was then done using cpDNA clones of Cryptomeria japonica and tobacco as probes. CpDNA variation was detected with enzyme-probe combinations: HindIII+pCS10 probe, HindIII+pCS7, and BglII+pCS7 in preliminary screening. These variations were considered to be caused by the same insertion, deletion or inversion. All populations surveyed for the combination HindIII+pCS10 resulted in only two frequency variations in each population. This indicates a gradual cline along latitude and longitude.

Detection of quantitative trait loci for juvenile growth, flower bearing and rooting ability based on a linkage map of sugi (Cryptomeria japonica D. Don)

Abstract Quantitative traits, including juvenile growth, flower bearing and rooting ability, of a woody plant species, Cryptomeria japonica D. Don, were analyzed in a three-generation pedigree with 73 F2 progenies using a linkage map with 85 genetic markers (72 RFLP, 11 RAPD, one isozyme and one morphological loci). A cluster of quantitative trait loci (QTLs) related to juvenile growth and female flower bearing was detected on linkage group 2. Some of the influence of this cluster could be attributed to pleiotropic effects of a dwarf locus located in its vicinity. QTLs related to male and female flower bearing were detected at different locations and showed different effects from each other, suggesting that the genetic systems controlling male and female flowering are different. No large QTL affecting rooting ability was detected in the material analyzed in this study.

In vitro regeneration of Alnus cremastogyne Burk from epicotyl explants.

SummaryMultiple shoots were grown from seedling explants of Alnus cremastogyne Burk by a two-stage culture procedure: initiation on WP medium supplemented with 2–8 μ M benzylammopurine(BAP) for 6 weeks, thereafter 3 weeks of subculture(shoot multiplication) on the same medium with 1 μ M BAP. A 5–9 fold multiplication rate was achieved. Type and concentration of sugar used in the multiplication medium were shown to be critical factors for both multiple shoot induction and bud elongation, the optima being 87.5mM glucose and 87.5mM sucrose respectively. After transfer to half-strength WP media either containing indolebutyric acid (IBA) or lacking plant growth regulator, almost all the shoots rooted. However, high rhizogenesis could be achieved only with shoots cultured in rooting medium containing 87.5mM sucrose or 175mM glucose, and shoots from multiplication media containing 87.5mM sucrose. Survival of the plantlets following transfer to vermiculite was 100%.

The Genetic Diversity of Isozymes and the Possible Dissemination of Ginkgo biloba in Ancient Times in Japan

Ginkgo biloba, a deciduous plant that was called a living fossil by Charles Darwin, is one of the most ancient gymnosperms in the world. Species of this genus were widely distributed throughout the world during the Jurassic period. Based on fossil records, it is estimated that at least 12 Ginkgo species existed. Most species of this genus and many other plant species perished during the glacial epochs of the Tertiary period. However, G. biloba is considered to have survived in southern China because of the warm climate. Natural Ginkgo trees are said to still exist in southern China. G. biloba has now spread by introduction to many countries worldwide as an ornamental tree.

Micropropagation of Bolaina Blanca (Guazuma crinita Mart.), a Fast-Growing Tree in the Amazon Region

Rapid clonal propagation of Bolaina blanca (Guazuma crinita Mart.) was established by the subculturing of the shoot-tips from aseptically germinated seedlings on woody plant medium (WPM) supplemented with trans-zeatin [trans-6-(4-Hydroxy-3-methylbut-2-enylamino)purine] (ZEA). After 45 days of culture, a seven-fold multiplication rate was achieved on WPM containing 10 μM of ZEA. Obtained shoots were simultaneously elongated and rooted on WPM containing 1 μM of kinetin [6-furfurylaminopurine] (KIN). After 60 days of culturing the growth of shoots was evident, and high rooting percentages were obtained. The plantlets were transferred into pots with vermiculite and acclimatized successfully in plastic boxes with transparent cover inside the growth cabinet.