Naoko Kitamoto

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.

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.

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.

Nine additional microsatellite markers for Primula sieboldii E. Morren

Saneyoshi Ueno*, Naoko Kitamoto, Ryo Ohsawa, Yoshihiko Tsumura & Izumi Washitani Genome Analysis Laboratory, Department of Forest Genetics, Forestry and Forest Products Research Institute, Tsukuba 305-8687, Japan; Laboratory of Plant Breeding, Institute of Life and Environmental Sciences, Tsukuba University, Tsukuba 305-8572, Japan; Laboratory of Conservation Ecology, Department of Ecosystem Studies, Institute of Agricultural and Life Sciences, Tokyo University, 1-1-1 Yayoi, Bunkyo-ku Tokyo, 311-8657, Japan (*Corresponding author: Phone: +81-298-73-3211; Fax: +81-298-74-3720; E-mail: saueno@ ffpri.affrc.go.jp)

QTL analysis of heterostyly in Primula sieboldii and its application for morph identification in wild populations

BACKGROUND AND AIMS Primula sieboldii is a perennial clonal herb that is distributed around the Sea of Japan and is endangered in Japan. Its breeding system is characterized by heteromorphic self-incompatibility, and the morph ratio within a population is very important for reproductive success. The aims of this study were to construct a linkage map, map the S locus as a qualitative trait and quantitative trait loci (QTLs) for floral morphological traits related to heterostyly, and predict the morph type in wild populations by using molecular markers for devising a conservation strategy. METHODS A linkage map was constructed with 126 markers. The QTLs for four floral traits and the S locus were mapped. Using the genotypes of loci that were located near both the S locus and the QTLs with large effects, morphs of 59 wild genets were predicted. KEY RESULTS The linkage map consisted of 14 linkage groups (LGs). The S locus was mapped to LG 7. Major QTLs for stigma and anther heights were detected in the same region as the S locus. These QTLs exhibited high logarithm of the odds scores and explained a high percentage of the phenotypic variance (>85 %). By analysing these two traits within each morph, additional QTLs for each trait were detected. Using the four loci linked to the S locus, the morphs of 43 genets in three wild populations could be predicted. CONCLUSIONS This is the first report of a linkage map and QTL analysis for floral morphology related to heterostyly in P. sieboldii. Floral morphologies related to heterostyly are controlled by the S locus in LG 7 and by several QTLs in other LGs. Additionally, this study showed that molecular markers are effective tools for investigating morph ratios in a population containing the non-flowering individuals or during the non-flowering seasons.

Development of 120 microsatellite markers for Primula sieboldii E. Morren for linkage mapping

Primula sieblodii E. Morren is a perennial herb, classified as ‘Near Threatened’ in the Japanese Red List. Using the magnetic enrichment method, we have developed 120 microsatellite markers and screened polymorphisms within the species as a whole as well as within a single population. At the species level, the average number of alleles per locus, and the average observed and expected heterozygosities were 7.06, 0.48, and 0.74, respectively; at the level of the single population these values were 5.53, 0.52, and 0.64, respectively. The microsatellite markers presented here will be valuable research tools in forthcoming projects, such as QTL mapping as well as for association studies of P. sieboldii.

Reconsideration for conservation units of wild Primula sieboldii in Japan based on adaptive diversity and molecular genetic diversity

Primula sieboldii E. Morren is a perennial clonal herb that is widely distributed in Japan, but in danger of extinction in the wild. In a previous study, we revealed the genetic diversity of the species using chloroplast and nuclear DNA and used this information to define conservation units. However, we lacked information on adaptive genetic diversity, which is important for long-term survival and, thus, for the definition of conservation units. In order to identify adaptive traits that showed adaptive differentiation among populations, we studied the genetic variation in six quantitative traits within and among populations for 3 years in a common garden using 110 genets from five natural populations from three regions of Japan. The number of days to bud initiation was adaptive quantitative trait for which the degree of genetic differentiation among populations (QST) was considerably larger than that in eight microsatellite markers (FST). The relationship between this trait and environmental factors revealed that the number of days to bud initiation was negatively correlated, with the mean temperature during the growing period at each habitat. This suggests that adaptive differentiation in the delay before bud initiation was caused by selective pressure resulting from temperature differences among habitats. Our results suggest that based on adaptive diversity and neutral genetic diversity, the Saitama population represents a new conservation unit.