Hideaki Taira

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

Abstract.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, Kendalls τ. 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.

Microsatellite markers reveal high allelic variation in natural populations of Cryptomeria japonica near refugial areas of the last glacial period

Using 11 microsatellite markers, we investigated the allelic variation and genetic structure of Cryptomeria japonica, across most of its natural distribution. The markers displayed high levels of polymorphism (average gene diversity=0.77, average number of alleles=24.0), in sharp contrast to the lower levels of polymorphism found in allozyme and cleaved amplified polymorphic sequence markers in previous studies. Little genetic differentiation was found among populations (FST=0.028, P<0.001), probably because the species is wind-pollinated and long-lived. No clear relationship between Nei’s genetic distances and geographical locations of the populations were found using the principal coordinate and unweighted pair-group method with arithmetic averaging analyses. The lack of such trends might be due partly to microsatellite homoplasy arising from mutation blurring the genealogical record. However, there was a trend towards high allelic diversity in five populations (Ashitaka, Ashiu, Oki-Island, Yakushima-Island-1 and -2), which are very close to, or in, refugial areas of the last glacial period as defined by Tsukada based on pollen analysis data and current climatic divisions. We postulate that these refugial populations might have been less affected by genetic drift than the other populations due to their relatively large size.

Gene flow and mating system in five Cryptomeria japonica D. Don seed orchards as revealed by analysis of microsatellite markers

We investigated gene flow in five Cryptomeria japonica D. Don seed orchards of two different types (common and miniature) at widely spaced locations using microsatellite markers. The quality of a seed crop is determined by many factors, including pollen contamination from outside sources, self-fertilization, and the proportion of contributions from constituent clones. Contamination rates were found to vary among ramets both within seed orchards (10.0–76.7% in the most variable seed orchard) and among seed orchards (35.0–65.8% on average). Among ramets, there were significant negative correlations between pollen contamination rate and their distance from the orchard edge; among seed orchards, there were significant positive correlations between the pollen contamination rate and the C. japonica forest area nearby. Some proportion of the pollen (10.7% of total contamination) also migrated from parts of the orchards that had not been treated with gibberellin to induce flowering. Self-fertilization rates varied among seed orchards (1.4–4.4% on average), and there were significant positive correlations between self-fertilization rate and the number of ramets per clone in both types of seed orchard. Contributions as pollen donors differed significantly among clones in all seed orchards. The distance between planted ramets, flowering phenology, and relative pollen fecundity may also have contributed to observed differences in paternal contribution. The influence of these factors on genetic potential did not differ greatly between the two types of orchards.

The construction of a high-density linkage map for identifying SNP markers that are tightly linked to a nuclear-recessive major gene for male sterility in Cryptomeria japonica D. Don

BackgroundHigh-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.ResultsWe 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.ConclusionsUsing 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.

Inheritance of the Trait of Male Sterility in Cryptomeria japonica

Matings of male-sterileC. japonica and fertile eliteC. japonica, as well as backcross seedlings of male-sterile trees, were carried out to clarify the genetic trait of male sterility of theC. japonica. The seeds from male-sterileC. japonica were germinated in an incubator and grown them in the greenhouse between 1994 and 1997. The seedlings were treated with 100 ppm gibberellin at early July 1994 and early July 1995 to promote the formation of male flowers. In the middle of January 1995, the male flowers of all seedlings were examined under the microscope to confirm the production of pollen. In January 1996, pollen did not developed in to the male flowers from the seedlings between the fertiled elite and backcrossC. japonica. In January 1997, all seedlings of eliteC. japonica produced pollen in their male flowers; however, pollen did not developed in 55 out of 120 backcrossed seedlings. This evidence suggests that the heredity pattern of male sterility inC. japonica is nuclear male sterility controlled by a pair of recessive genes.

Cytological and Genetical Studies on Male Sterility in Cryptomeria japonica D. Don

Genetic male sterility is a useful trait in plant breeding, especially in angiosperm crops such as corn, onion and carrot. We found a male sterile sugi (Cryptomeria japonica D. Don) tree in Toyama, Japan. Pollen of sugi is one of the major causes of pollinosis in Japan. We carried out this research in an attempt to make clear the characteristics and inheritance of this male sterility. Microsporogenesis of the male sterile tree proceeded meiosis, however, the microspores collapsed after they were separated from pollen tetrads in locules, resulting in complete male sterility. Most likely, ethylene evolution was responsible for male sterility expression. Full seed setting in the male sterile tree indicated normal macrosporogenesis. Seeds obtained from crossing between male sterile and normal lines showed relatively high level of germination and their seedlings grew vigorously. The somatic chromosome numbers of 241 germinated seeds, derived from the male sterile tree, were mostly 22, euploid. These results indicated that male sterile tree was different from other similar previously reported trees with low pollen fertility, resulting from triploid or trisomics. Probably, male sterility in sugi is either nuclear genetic male sterility or cytoplasmic male sterility.

Factors Influencing Male Reproductive Success in a Cryptomeria japonica Seed Orchard Revealed by Microsatellite Marker Analysis

Abstract We investigated the influence of male flower production, floral synchrony and inter-tree distances on male reproductive success in a miniature seed orchard of Cryptomeria japonica. We used six microsatellite markers to determine the paternity of each seed. In the seed orchard, the average pollen contamination and clonal self-fertilization rates were 38.7% and 1.7%, respectively. The level of male reproductive success of constituent clones varied from 0.0 to 15.7%. Five clones showing the highest male reproductive success contributed ca. 30% of all analyzed seeds as a pollen donor after excluding contamination by external sources of pollen. The statistical analyses showed that male reproductive success was strongly influenced by male flower production of each clone and, possibly, by their distance to the mother trees. The linear regression which included male flower production and floral synchrony as independent variables, however, accounted for only 14.7% of variation of male reproductive success, suggesting that other factors such as pollen competition might also influence male reproductive success. Since we found no significant correlation between male reproductive and female reproductive successes, it may be better to equalize male and female reproductive successes independently

A histological comparison of the development of pollen and female gametophytes in fertile and sterile Cryptomeria japonica

To determine a possible mechanism causing male and female sterility in Cryptomeria japonica male and female cones were collected from a C. japonica, tree, ShinDai2, that lacks pollen release and fertile seeds and specimens were processed to examine the development of pollen and female gametophytes using light microscopy and field emission scanning electron microscopy. Pre-meiotic development proceeded normally, but the formation of aberrant meiotic products was observed in cones of both sexes. In sterile microsporangia, heterogeneous microspore populations ranging from monads to polyads gave rise to mature pollen grains of non-uniform size. These pollen grains were covered with an amorphous layer and adhered to each other. In addition, they remained in the microsporangia and were not released even after the onset of pollen dissemination from fertile trees. In the ovules of sterile female cones, megaspores with abnormal shapes, numbers, and sizes formed, and the development of female gametophytes was arrested at the free nuclear or archegonium formation stages. These gametophytes collapsed, and no fertile embryo was generated. Results indicate that meiotic defects are important in the sterility mechanism.

Stand structure and composition of a 60-year-old larch (Larix kaempferi) plantation with retained hardwoods

Tree retention is understood as a key practice in creating complexity, leading to heterogeneity in resources and habitats in managed stands. In this article, we clarify the long-term effects of tree retention on stand structure and tree-species composition in a 60-year-old Larix kaempferi plantation in central Japan. In our study plot (1.5 ha) there were 18 stems/ha of retained trees (determined by tree-ring analysis), mostly Quercus crispula. We conducted spatial analyses and tested the hypothesis that tree abundance, size structure, and species composition and diversity change with distance from the retained trees. Near the retained trees, L. kaempferi showed a reduction of 40%–60% in basal area, due presumably to the shading effect. In contrast, the nearby area showed greater species diversity in the canopy layer. The retained trees created patches of different species composition in the understory. The spatial gradient of shade and colonization opportunity provided by retained trees greatly affect the distribution of the colonized species, according to their shade tolerance and seed-dispersal ability, which resulted in the stand structure with a heterogeneous shrub-layer vegetation. Retention proved particularly important for the enhancement and long-term maintenance of structural and compositional complexity in L. kaempferi plantations.

Plus tree of Cryptomeria japonica D. Don with a heterozygous male-sterility gene

To find plus tree clones of Cryptomeria japonica that are heterozygous for a male-sterility gene (Aa), we crossed a homozygous male-sterile tree (aa) with 63 clones. Male sterility in this case is controlled by a recessive allele at a single gene locus and is expressed only in homozygotes. All F1 seedlings obtained by crossing the male-sterile mother tree and 62 out of the 63 clones produced pollen. In contrast, F1 seedlings obtained from the crossing between the male-sterile mother tree and a plus tree clone, Ohara 13, produced 64 male-sterile individuals and 52 fertile individuals. The segregation ratio fitted the expected 1 : 1 ratio according to a chi-square test. These results clearly demonstrate that the Ohara 13 clone is heterozygous for a male-sterility gene.