Masanori Honjo

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.

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.

Strawberry cultivar identification based on hypervariable SSR markers

We genotyped strawberry cultivars by two newly selected and two previously reported SSR markers. All four markers produced interpretable electropherograms from 75 accessions consisting of 72 Fragaria × ananassa cultivars or lines and three octoploid Fragaria species accessions. These SSR markers were highly polymorphic; in particular, one of the newly developed markers, FxaHGA02P13, was capable of distinguishing all of the accessions except for a mutant strain that was derived from another accession in the set. When two markers were combined, all 48 full-sib individuals could be distinguished. Fingerprinting patterns were reproducible between multiple samples, including the leaves, sepals, and fruit flesh of the same accession. Principal-coordinate analysis of the 75 accessions detected several groups, which reflect taxon and breeding site. Together with other available markers, these SSR markers will contribute to the management of strawberry genetic resources and the protection of breeders’ rights.

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.

Origins of traditional cultivars of Primula sieboldii revealed by nuclear microsatellite and chloroplast DNA variations

We examined the origins of 120 cultivars of Primula sieboldii, a popular Japanese pot plant with a cultivation history of more than 300 years. In an assignment test based on the microsatellite allelic composition of representative wild populations of P. sieboldii from the Hokkaido to Kyushu regions of Japan, most cultivars showed the highest likelihood of derivation from wild populations in the Arakawa River floodplain. Chloroplast DNA haplotypes of cultivars also suggested that most cultivars have come from genets originating in wild populations from the same area, but, in addition, that several are descended from genets originating in other regions. The existence of three haplotypes that have not been found in current wild populations suggests that traditional cultivars may retain genetic diversity lost from wild populations.