Asako Matsumoto

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.

Amounts and patterns of nucleotide variation within and between two Japanese conifers, sugi (Cryptomeria japonica) and hinoki (Chamaecyparis obtusa) (Cupressaceae sensu lato)

Sugi (Cryptomeria japonica) and hinoki (Chamaecyparis obtusa) are the most important timber species in Japan. To quantify and compare the level of nucleotide variation in these species, we investigated their variation at ten nuclear loci. Average values of nucleotide diversity at synonymous sites (πSYN) found in sugi and hinoki were 0.0038 and 0.0069, respectively. However, although the average value of nucleotide diversity was higher in hinoki than in sugi, their average values of haplotype diversity were similar. Deviations from the standard neutral model were detected at two loci in hinoki using Tajima’s D, Fay and Wu’s H, and Strobeck’s S statistics, which seem to be due to its historical population structure. Levels of divergence between the two species at synonymous sites of the ten genes ranged from 0.121 to 0.566 (0.28 on average). These values positively correlated with their guanine + cytosine contents at third-codon positions of synonymous sites (%GC3s).

Characterization of EST–SSRs from Cryptomeria japonica

Simple sequence repeat (SSR) markers were developed for Cryptomeria japonica from 55,530 expressed sequence tags (ESTs). Of the 219 designed primer pairs, 176 showed clear PCR amplification products and 27 of these revealed polymorphisms in six individuals sampled at sites across Japan. When these 27 loci were examined in 48 individuals from a single population, the number of alleles and the Polymorphism Information Content (PIC) ranged from 2 to 17 and 0.09 to 0.86, respectively. The average PIC value obtained from EST–SSR markers (0.40) was lower than that from genomic SSR markers (0.67 and 0.62). We observed a significant departure from Hardy–Weinberg equilibrium in five of the 27 loci. These EST–SSR markers will be useful for the evaluation of genetic variation in C. japonica and for genetic mapping.

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.

Clone identification in Japanese flowering cherry (Prunus subgenus Cerasus) cultivars using nuclear SSR markers

Numerous cultivars of Japanese flowering cherry (Prunus subgenus Cerasus) are recognized, but in many cases they are difficult to distinguish morphologically. Therefore, we evaluated the clonal status of 215 designated cultivars using 17 SSR markers. More than half the cultivars were morphologically distinct and had unique genotypes. However, 22 cultivars were found to consist of multiple clones, which probably originate from the chance seedlings, suggesting that their unique characteristics have not been maintained through propagation by grafting alone. We also identified 23 groups consisting of two or more cultivars with identical genotypes. Most members of these groups were putatively synonymously related and morphologically identical. However, some of them were probably derived from bud sport mutants and had distinct morphologies. SSR marker analysis provided useful insights into the clonal status of the examined Japanese flowering cherry cultivars and proved to be a useful tool for cultivar characterization.

Development and characterization of 10 microsatellite markers from Wisteria floribunda (Fabaceae)

Abstract Wisteria floribunda is a deciduous liana species widely distributed in Japan. It is essential to evaluate the clonal structure of this species because high clonal ability enables lianas to make dense colonies after canopy gaps occur. Therefore, we developed 10 microsatellite markers for W. floribunda from genomic shotgun sequences available in the publicly available database to study clonal structure and life history strategy of this species. The number of alleles per locus ranged from 7 to 16, with an average of 10.2. The observed and expected heterozygosity ranged from 0.483 to 0.926 and from 0.642 to 0.916, with averages of 0.769 and 0.800, respectively. These findings will contribute to the understanding of ecological roles and management of this species.

Large contribution of clonal reproduction to the distribution of deciduous liana species (Wisteria floribunda) in an old-growth cool temperate forest: evidence from genetic analysis

Background and Aims Extensive clonal (vegetative) reproduction in lianas is a common and important life history strategy for regeneration and colonization success. However, few studies have evaluated the contribution of clonal reproduction to stand-level distribution of lianas in their natural habitat using genetic tools. The objectives of the present study were to investigate (1) the contribution of clonal reproduction to the distribution of Wisteria floribunda, (2) the size of clonal patches and (3) how the distribution patterns of W. floribunda clones are affected by micro-topography. Methods The contribution of clonal reproduction to the distribution of the deciduous liana species W. floribunda was evaluated using genetic analysis across a 6-ha plot of an old-growth temperate forest in Japan and preference in landform between clonal ramets and non-clonal ramets was assessed. Key Results Of the 391 ramets sampled, clonal reproduction contributed to 71 and 62 % of the total abundance and basal area, respectively, or 57 and 31 % when the largest ramet within a genet was excluded. The large contribution of clonal reproduction to the density and basal area of W. floribunda was consistent with previous observational studies. The largest genet included a patch size of 0.47 ha and ranged over 180 m. Preferred landforms of clonal and non-clonal ramets were significantly different when evaluated by both abundance and basal area. Non-clonal ramets distributed more on lower part of the slope than other landforms in comparison with clonal ramets and trees, possibly reflecting the limitation of clonal growth by stolons. Conclusions Using genetic analysis, the present study found evidence of a large contribution of clonal reproduction on the distribution of W. floribunda in its natural habitat. The results indicate that clonal reproduction plays an important role not only in the formation of populations but also in determining the distribution patterns of liana species.

Evidence of clonal propagation in Cryptomeria japonica D. Don distributed on Pacific Ocean side in Japan

Abstract Cryptomeria japonica is distributed on the Pacific Ocean side and reproduces only by seedling; however, C. japonica var. radicans is distributed on the Japan Sea side and reproduces by both seedling and layering. Until now, there has been no report that C. japonica on the Pacific Ocean side regenerates by layering. In this study, the regeneration system of C. japonica in a national forest of Miyagi Prefecture Ishinomaki City was investigated using DNA analysis. C. japonica growing in the study plot was divided into seven genets and 40 single ramets, clearly suggesting that C. japonica at this site regenerates not only by seedling, but also by layering. The strong winds and low temperature appear to be key factors in layering reproduction at this site.

Isolation and characterization of microsatellite markers from the RAD sequence of two temperate liana species: Euonymus fortunei (Celastraceae) and Schizophragma hydrangeoides (Hydrangeaceae)

Abstract Clonal reproduction of lianas is a common but important life history strategy. It is necessary to evaluate the clonal structure of liana species because clonal ability is potentially a major determinants distribution pattern of lianas. Therefore, we developed 10 microsatellite markers for Euonymus fortunei and Schizophragma hydrangeoides respectively from genomic sequences obtained from double-digest restriction site associated DNA (ddRAD). The sequence data of the developed markers were deposited on the public database. The expected heterozygosity (HE) of E. fortunei and S. hydrangeoides ranged from 0.727 to 0.847 with an average of 0.766, and from 0.734 to 0.924 with an average of 0.812, respectively. All loci were under HWE except for a locus of S. hydrangeoides (sh07). These markers should contribute to the understanding of the life history of temperate liana species.