Ryo Ohsawa

Spatial genetic structure among and within populations of Primula sieboldii growing beside separate streams

We investigated the hierarchical genetic structure of SSR (simple sequence repeats) and cpDNA (chloroplast DNA) polymorphisms among and within populations of Primula sieboldii, a heterostylous clonal herb. Seven out of eight populations at the study site, located in a mountainous region of Nagano Prefecture, had each developed alongside a different stream, and the other occurred on a flat area 70 m from the nearest stream. The magnitude of genetic differentiation among streamside populations in maternally inherited cpDNA (Φ = 0.341) was much higher than that in biparentally inherited SSRs (Φ = 0.011). This result suggests that seed dispersal among streams was restricted, and pollen was the primary agent of gene flow among streamside populations. In contrast, genetic differentiation among subpopulations within streams were low at both markers (Φ = 0.053 for cpDNA, Φ = 0.025 for SSR). This low differentiation among subpopulations in cpDNA compared with that among streamside populations suggest that seed dispersal occur along the stream probably during flooding. This hypothesis was supported by the fact that in cpDNA haplotypes, no clear genetic structure was detected within the streamside population, while a significant genetic structure was found within 20 m in the nonstreamside population. Furthermore, within the streamside populations, two pairs of ramets with identical multilocus genotypes for eight SSR loci were distantly (> 50 m) distributed along the same streamside, suggesting dispersal of clonal propagule. Our study showed that the heterogeneity of the landscape can influence gene flow and hence spatial genetic structure.

Mapping QTLs influencing rice floral morphology using recombinant inbred lines derived from a cross between Oryza sativa L. and Oryza rufipogon Griff.

Abstract. To understand the genetic basis of floral traits associated with the mating system in rice, we analyzed pistil, stamen and glume traits using a recombinant inbred line population, derived from a cross between an Asian cultivated rice (Oryza sativa L.), Pei-kuh, and a wild rice (Oryza rufipogon Griff.), W1944. Quantitative trait loci (QTLs) affecting floral morphology were detected by composite interval mapping using a linkage map constructed using 147 markers, mostly RFLPs. A total of 7, 4, 14 and 6 QTLs were detected for traits related to pistil, stamen, and size and shape of the glume, respectively. Comparison of 31 QTLs affecting these organs revealed ten QTLs affecting the different organs in four adjacent regions on chromosomes 2, 4, 5 and 10, but most QTLs (68%) were located separately on the whole chromosomes. Although four QTLs for stigma breadth, anther length and thickness of lemma and palea explained more than 25% of the total phenotypic variance, most QTLs (87%) had smaller effects. These results suggest that quantitative variation observed for pistil, stamen and glume traits is controlled by several distinct genes with small effects.

Quantitative evaluation of common buckwheat (Fagopyrum esculentum Moench) kernel shape by elliptic Fourier descriptor

Kernel shape of 27 common buckwheat cultivars was evaluated quantitatively by image analysis using elliptic Fourier descriptors and their principal components. The relationships between the quantitative information on kernel shape and several agronomic characteristics were clarified. The closed contour of each kernel projection was extracted, and 80 elliptic Fourier coefficients were calculated for each contour. The Fourier coefficients were standardized so that they were invariant of the size, rotation, shift and chain-code starting point. Then, the principal components on the standardized Fourier coefficients were evaluated. The cumulative contribution at the fifth principal component was higher than 93%. It was found that the first, second, third and fifth principal components represented the aspect ratio of the kernel, the location of the kernel centroid, the sharpness of the two kernel tips and the roundness of the base in the kernel contour, respectively. Analysis of the variance indicated that genotypic differences in these principal components were significantly large. Because these variations of the principal components were continuous, the effect of polygenes on shape was suggested. The relationships between these principal components and agronomic traits, including flowering period, yield, 1000 seed weight and volume weight, were also investigated. It was clarified that the first principal component was closely correlated with agronomic traits such as flowerig period, 1000 seed weight and volume weight. In conclusion, it was clarified that principal component scores based on elliptic Fourier descriptors yield useful quantitative parameters for evaluating kernel shape for common buckwheat breeding.

Genetic structure of landraces in foxtail millet (Setaria italica (L.) P. Beauv.) revealed with transposon display and interpretation to crop evolution of foxtail millet

Although the origin and domestication process of foxtail millet (Setaria italica subsp. italica (L.) P. Beauv.) has been studied by several groups, the issue is still ambiguous. It is essential to resolve this issue by studying a large number of accessions with sufficient markers covering the entire genome. Genetic structures were analyzed by transposon display (TD) using 425 accessions of foxtail millet and 12 of the wild ancestor green foxtail (Setaria italica subsp. viridis (L.) P. Beauv.). We used three recently active transposons (TSI-1, TSI-7, and TSI-10) as genome-wide markers and succeeded in demonstrating geographical structures of the foxtail millet. A neighbor-joining dendrogram based on TD grouped the foxtail millet accessions into eight major clusters, each of which consisted of accessions collected from adjacent geographical areas. Eleven out of 12 green foxtail accessions were grouped separately from the clusters of foxtail millet. These results indicated strong regional differentiations and a long history of cultivation in each region. Furthermore, we discuss the relationship between foxtail millet and green foxtail and suggest a monophyletic origin of foxtail millet domestication.

Effect of flowering phenology on pollen flow distance and the consequences for spatial genetic structure within a population of Primula sieboldii (primulaceae)

To evaluate the effects of flowering phenology on pollen flow distance and spatial genetic structure in a population of a bumblebee-pollinated herb, Primula sieboldii, we investigated the flowering phenology of 1712 flowers of 97 genets in a population in Nagano Prefecture, Japan, and constructed a mating model based on the observed mating pattern, which was revealed by paternity analysis using 11 microsatellite markers. The effects of flowering phenology were inferred by comparing estimated pollen flow distance and the level of heterozygosity in the next generation between two scenarios. In the first scenario, both the intergenet distance and flowering phenology influenced mating opportunity, while in the second scenario only intergenet distance influenced mating opportunity. Although the frequency distribution of pollen flow distance at the population level did not differ significantly between the two scenarios, the mean pollen flow distance of several flowers increased by more than 10 m as a result of variation in flowering phenology. Furthermore, accounting for flowering phenology predicted change in heterozygosity in the next generation from -0.04 to 0.07. The results showed that flowering phenology can affect pollen flow distance and spatial genetic structure.

Inter-varietal variations of rutin content in common buckwheat flour (Fagopyrum esculentum Moench.)

SummaryTo improve the quality of buckwheat flour, we investigated the effect of the cropping season on the rutin content in various cultivars of common buckwheat (Fagopyrum esculentum Moench.). Thirty cultivars of buckwheat were grown under both long day conditions (the summer cropping) and short day conditions (the late summer cropping). The inter-varietal variations and the effect of the cropping season on the rutin content were examined. The rutin content was determined by high performance liquid chromatography (HPLC). Results show that the average rutin content in the late summer cropping was about half that of the summer cropping. There were wide inter-varietal variations of rutin content in the summer cropping. The differences in rutin content in the late summer cropping were less pronounced. Rutin content was closely correlated with the flowering period in summer (r=0.735***). The later flowering cultivars produced a higher content of rutin than the earlier flowering cultivars in the summer cropping. The differences in the cumulative solar radiation received in the two seasons seems to be a reasonable explanation for this increase in the rutin content of the buckwheat flour. The rutin content of early flowering cultivars, which are suitable for long day conditions, is positively correlated with yield in the summer cropping. However, the rutin content in the intermediate and late summer ecotype cultivars, which are suitable for short day conditions, shows no correlation with yield in the late-summer cropping. In conclusion, there are wide inter-varietal variations of rutin content in buckwheat flour and the rutin content increases under long day conditions.

Quantitative evaluation of the petal shape variation in Primula sieboldii caused by breeding process in the last 300 years

Primula sieboldii (E. Morren) has been a popular garden plant at least since the Edo period, about 300 years ago. We compared petal form between cultivars and wild populations in order to characterise the changes that have occurred during domestication. The comparison was made using EF-PCA analysis, which describes overall petal shape mathematically by transforming petal contour coordinates into elliptic Fourier descriptors; it subsequently summarises these descriptors by principal component analysis (PCA). Rearing cultivars in a common-garden experiment identified the PCs with a substantial genetic element. A clear heritable component was detected for the PCs characterising symmetrical variation in flower shape, but not the asymmetrical variation. Wild populations of this species have become endangered owing to habitat destruction by human activity, and many lowland floodplain habitats have been lost. Variation within the remaining wild populations was significantly lower than in the cultivars for PC1 (aspect ratio), PC3 (curvature of proximal and distal parts) and petal area; but not for PC2 (depth of head notch) and PC4 (position of the centre of gravity). The shifts in petal form from the wild populations to the cultivars parallel those seen in other crop-types following domestication, including an increase in size and diversity of forms: cultivars have shallower head notches, more fan-shaped petals and larger petals than do wild P. sieboldii.

Management units of the endangered herb Primula sieboldii based on microsatellite variation among and within populations throughout Japan

To promote programs for the conservation and restoration of the endangered species Primula sieboldii, we examined genetic variation at eight microsatellite loci among and within 32 remnant wild populations throughout Japan. Total allelic diversity within a population was higher in larger populations, but not so after rarefaction adjustment. The positive relationship between population size and the inbreeding coefficient may suggest that more heterozygous genets tend to survive the habitat contraction possibly because of the higher fitness associated with heterozygosity. By principal coordinate analysis and Bayesian analysis, we detected four genetic groups (Hokkaido, northern Honshu, central Honshu, and western Japan), which could be recognized as management units of P. sieboldii. If supplementation with plants from other populations were planned, it should be conducted among populations which belong to the same management unit and which are likely to represent the same adaptive variation.

Quantitative evaluation of flower colour pattern by image analysis and principal component analysis of Primula sieboldii E. Morren

In Primula sieboldii E. Morren, many cultivars have been bred with the purpose of obtaining various petal colour patterns. Colour pattern is an important breeding target in this species, and the availability of an objective and quantitative evaluation method is of vital importance for both genetic analysis and variety registration. Our objective was to establish a new quantitative evaluation method of P. sieboldii flower colour patterns in regions of interest (ROIs) by principal component analysis (PCA). We first set a ROI in each petal as a region that represented the petal colour pattern and defined the maximum square on each petal as the ROI. We then converted each ROI image to a 10 × 10 pixel mosaic image and defined a total of 300 variables (the colour values for red, green and blue components of the 100 pixels) per mosaic image. Finally, we summarized the information on the 300 variables by PCA, and redrew the mosaic images to correspond to some typical principal component scores to determine the effect of each principal component on colour pattern. By this method, we detected five different features of petal colour pattern, four of which were revealed to be mainly genetically controlled. Thus, we successfully established a procedure for evaluating petal colour patterns in P. sieboldii cultivars. This new procedure can be used as a basis for an objective and efficient variety registration system.

Effect of floral morphology on pollination in Brassica rapa L

SummaryFlower structure, especially the anther–stigma separation (ASS), is well known to affect pollination efficiency, and thus to potentially increase or decrease seed production in crops. Therefore, investigating the relationship between flower characteristics and pollination ability is crucial to a full understanding of mechanisms to improve F1 seed production in Brassica rapa. We used image analysis to measure three flower characteristics: short stamen height (SSH); long stamen height (LSH); and pistil height (PH) in seven cultivars. We calculated the ratio of PH to LSH as an index of anther–stigma separation (ASS). We investigated the number of pollen grains (NPG) deposited on the stigma and the seed-set percentage (SSP) under open-pollination and self-pollination conditions (with and without insects, respectively). Nested ANOVA indicated significant differences between the seven cultivars in the floral characteristics except for PH. Moreover, much larger variation was observed in NPG and SSP than in floral characteristics. Although stepwise multiple regression analysis indicated that plants with relatively high PHs produced more seed under self-pollination, the number of seeds that resulted from self-pollination did not affect seed production because of incompatibility. Therefore, the effect of the spatial position of pistils on the F1 seed production was low. Possibly other factors such as the total pollen production and visiting times of pollinators were important factors in the low yields observed in some cultivars