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In apple (Malus spp.), crown gall disease, caused by the bacterial pathogens Rhizobium radiobacter (Ti) and R. rhizogenes (Ti), can be severe. To control the disease, breeding of apple rootstocks that exhibit crown gall resistance is a promising approach. In this study, we used a full-sib F1 population derived from a ‘JM7’ (susceptible) × Sanashi 63 (resistant) cross to identify quantitative trait loci (QTLs) that control resistance to tumour-inducing Rhizobium isolates found in apple production areas in Japan. Three stable QTLs, associated with a wide range of crown gall resistances, were identified in three linkage groups (LGs). QTLs for resistance to isolates Peach CG8331, Nagano 1 and Nagano 2 co-localised at the middle of LG 2 in Sanashi 63, where the crown gall resistance gene Cg (renamed as Rrr1 in this study) was previously identified. Similarly, in ‘JM7’, QTLs were identified on LG 11 for resistance to isolates ARAT-001, ARAT-002 and Kazuno 2, and on LG 15 for resistance to isolate ARAT-001. Fine-mapping of Rrr1 and nucleotide sequencing of a contig obtained from two bacterial artificial chromosome (BAC) clones delimited the Rrr1 gene to a region spanning 217 kb. In silico gene prediction from the region identified four genes encoding the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class plant resistance genes.

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We developed new F1 hybrids of Chinese cabbage (Brassica rapa L. ssp. pekinensis) that allow cultivation earlier in spring without heating by introducing extremely late-bolting alleles at two homologs of the flowering repressor Flowering Locus C (BrFLC2 and BrFLC3) from non-heading ‘Leafy Green Parental Line No. 2’. These new F1 hybrids were produced by the following four steps. First, the extremely late bolting selected lines were developed. These selected lines headed in spring after overwintering cultivation, whereas the conventional F1 cultivars flowered. Secondly, an investigation of the three plantings showed that our F1 hybrids formed heads when seeds were sown from mid-February to early March, whereas the conventional F1 cultivar did not form heads because of premature bolting. Thirdly, we identified some F1 hybrids with extremely late bolting during early spring cultivation in an investigation of many F1 hybrids. Finally, based on an investigation across four cold regions for 2 years, we compared the commercialization rate, defined as the proportion of plants greater than 2000 g in weight and with a flowering stalk less than 10 cm long. Then we identified a F1 of MS02 × 12-04 which had a high commercialization rate on average (92%), whereas the rates of three conventional F1 cultivars were only 0–2%. In the near future, these F1 hybrids will be valuable late-bolting cultivars despite climate change, permitting stable cultivation and harvest over wide regions.

†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.

Primula sieboldii is a self-incompatible ornamental plant that has been cultivated for more than 300 years in Japan. In order to increase the available genomic resources for this species, 5651 expressed sequence tags (ESTs) were generated from seedling and winter bud cDNA libraries. After clustering and assembling ESTs, 2960 unigene elements that contain an homologous sequence to an S locus-linked gene of P. vulgaris were identified. In total, 127 simple sequence repeats (SSRs) were found. The most frequent di- and tri-SSR motifs were GA (78.6%) and ATT (12.9%), respectively. Twenty SSR and 40 single nucleotide polymorphism (SNP) markers were developed. These markers were evaluated for polymorphisms using 24 individuals (Y) from a population in Yatsugatake and 14 individuals (PS) from across the distributional range of P. sieboldii in Japan. The average level of expected heterozygosity within Y and PS was 0.39 and 0.48, respectively for EST-SSR markers and 0.26 and 0.29, respectively for SNP markers. The level of polymorphism for EST-SSR markers was lower than that for genomic SSR markers developed in a previous study. We also surveyed the genetic diversity of 56 cultivars, which had lower allelic richness than the Y and PS populations.

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.

Radish (Raphanus sativus L.) is a useful vegetable with diverse features and worldwide distribution; however, the diversification and domestication history of cultivated radish has not been well documented. In order to understand genetic relationships among radishes around the world and the resulting diversity, we analyzed 65 accessions of cultivated radish collected from 21 Eurasian and North African countries using 221 amplified fragment-length polymorphism (AFLP) markers. These accessions formed four groups according to their provenance (Europe, Middle East, South Asia, and East Asia) in a neighbor-joining (NJ) tree. Despite geographical barriers, there might thus be a frequent exchange of germplasms within each region. The average genetic diversity did not differ significantly among the groups, ranging from 0.267 (Middle East) to 0.297 (East Asia), indicating that no obvious bottleneck effect in each region has occurred during the spread of this species.

Primula sieboldii (E. Morren), the ancestor of the Japanese garden Primula, is in danger of extinction in the wild. Genetic diversity is a key component for conservation efforts associated with population management. Genetic diversity in visible traits and several molecular makers were evaluated, respectively. Since it is difficult to determine the degree to which genetic and environmental differences contribute to observed phenotypic variation in natural habitats. A common-garden approach is used. The contribution of genetic and environmental differences to variation in petal shape and area of Primula sieboldii were evaluated. Samples from 108 genets gathered from five natural populations in three regions of Japan were analyzed in a common-garden experiment and also analyzed using eight microsatellite markers. From the results of quantitative evaluation based on image analysis, broad genetic variation in petal traits within populations and low level of population differentiation was found. For all petal shapes, Qst was smaller than Fst, suggesting that wild populations might be under moderate selective pressure for a specific phenotype. For petal area, Qst was nearly equal to Fst, suggesting that population differentiation has been caused mainly by genetic drift.

We investigated the effect of population density of compatible neighbours on inbreeding level of Primula sieboldii, a heterostylous clonal herb. Pollinator availability, seed set, selfing rate, diversity of pollen donors, and fitness of progenies were compared between less and more isolated genets, which differed in the number of compatible opposite-morph genets within 20 m, the range at which most pollen flow occurred. Although pollinator availability did not differ between the two groups, seed set and diversity of pollen donors in more isolated genets were significantly lower than in less isolated genets. Additionally, the mean selfing rates of less and more isolated genets were 1.3 and 36.7%, respectively, and the mean leaf area of the self-fertilized seedlings was 70 to 40% smaller than that of outcrossed seedlings of the same mother genet. Due to this large inbreeding depression, it is unlikely that self-fertilized seedlings could successfully establish in natural habitats and hence the inbreeding level in the next generation around the more isolated genet would not increase rapidly. However, the possibility of mating between full-sibs would increase because the diversity of pollen donors was low and both pollen and seed dispersal were spatially restricted. Thus the inbreeding level of the next generations would gradually increase around the more isolated genets owing to biparental inbreeding. This study suggested that the population density of compatible neighbours has a critical impact on the future inbreeding level within P. sieboldii populations.

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.

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 Φ 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 Φ Φ = 0.053 for cpDNA, Φ Φ 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.

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.

With the proliferation of genetically modified (GM) products and the almost exponential growth of land use for GM crops, there is a growing need to develop quantitative approaches to estimating the risk of escape of transgenes into wild populations of crop relatives by natural hybridization. We assessed the risk of transgene escape by constructing a population genetic model based on information on fitness-related QTLs obtained from an F 2 population of wild soybean G. soja × cultivated soybean Glycine max. Simulation started with ten F 1 and 990 wild soybeans reproducing by selfing or outcrossing. Seed production was determined from the genetic effects of two QTLs for number of seeds (SN). Each seed survived winter according to the maternal genotype at three QTLs for winter survival (WS). We assumed that one neutral transgene was inserted at various sites and calculated its extinction rate. The presence of G. max alleles at SN and WS QTLs significantly decreased the probability of introgression of the neutral transgene at all insertion sites equally. The presence of G. max alleles at WS QTLs lowered the risk more than their presence at SN QTLs. Although most model studies have concentrated only on genotypic effects of transgenes, we show that the presence of fitness-related domestication genes has a large effect on the risk of transgene escape. Our model offers the advantage of considering the effects of both domestication genes and a transgene, and they can be widely applied to other wild × crop relative complexes.

SummaryPrimula sieboldiiE. 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.

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)