Genome-wide association study of individual sugar content in fruit of Japanese pear (Pyrus spp.).

BACKGROUND: Understanding mechanisms of sugar accumulation and composition is essential to determining fruit quality and maintaining a desirable balance of sugars in plant storage organs. The major sugars in mature Rosaceae fruits are sucrose, fructose, glucose, and sorbitol. Among these, sucrose and fructose have high sweetness, whereas glucose and sorbitol have low sweetness. Japanese pear has extensive variation in individual sugar contents in mature fruit. Increasing total sugar content and that of individual high-sweetness sugars is a major target of breeding programs. The objective of this study was to identify quantitative trait loci (QTLs) associated with fruit traits including individual sugar accumulation, to infer the candidate genes underlying the QTLs, and to assess the potential of genomic selection for breeding pear fruit traits. RESULTS: We evaluated 10 fruit traits and conducted genome-wide association studies (GWAS) for 106 cultivars and 17 breeding populations (1112 F1 individuals) using 3484 tag single-nucleotide polymorphisms (SNPs). By implementing a mixed linear model and a Bayesian multiple-QTL model in GWAS, 56 SNPs associated with fruit traits were identified. In particular, a SNP located close to acid invertase gene PPAIV3 on chromosome 7 and a newly identified SNP on chromosome 11 had quite large effects on accumulation of sucrose and glucose, respectively. We used 'Golden Delicious' doubled haploid 13 (GDDH13), an apple reference genome, to infer the candidate genes for the identified SNPs. In the region flanking the SNP on chromosome 11, there is a tandem repeat of early responsive to dehydration (ERD6)-like sugar transporter genes that might play a role in the phenotypes observed. CONCLUSIONS: SNPs associated with individual sugar accumulation were newly identified at several loci, and candidate genes underlying QTLs were inferred using advanced apple genome information. The candidate genes for the QTLs are conserved across Pyrinae genomes, which will be useful for further fruit quality studies in Rosaceae. The accuracies of genomic selection for sucrose, fructose, and glucose with genomic best linear unbiased prediction (GBLUP) were relatively high (0.67-0.75), suggesting that it would be possible to select individuals having high-sweetness fruit with high sucrose and fructose contents and low glucose content.

Development of an SSR marker set for efficient selection for resistance to black spot disease in pear breeding.

Black spot disease, which is caused by Alternaria alternata (Fries) Keissler Japanese pear pathotype, is one of the most harmful diseases in Japanese pear cultivation. Because of the potential harm of fungicides to consumers and the environment, resistant cultivars are desired. In this study, to enable efficient marker-assisted selection in pear breeding, we conducted comprehensive inoculation tests and genotyping with 207 pear cultivars. We identified a marker set (Mdo.chr11.27 and Mdo.chr11.34) suitable for selection for black spot resistance. In most susceptible cultivars, Mdo.chr11.27 amplified a 220-bp band and Mdo.chr11.34 amplified a 259-bp band. The genotype of Mdo.chr11.34 corresponds perfectly to the estimated genotype of Japanese pears susceptible to black spot disease. Using linkage analysis, we identified the positions of the gene for susceptibility to black spot disease in Chinese pear. Mdo.chr11.27 and Mdo.chr11.34 were tightly linked to susceptibility in Chinese pear, and the susceptibility gene was mapped at the top of linkage group 11, similar to that in Japanese pear. This marker set and the accumulation of phenotypic data will enable efficient marker-assisted breeding for black spot resistance in pear breeding.

SSR markers developed using next-generation sequencing technology in pineapple, Ananas comosus (L.) Merr.

Simple sequence repeat (SSR) markers provide a reliable tool for the identification of accessions and the construction of genetic linkage maps because of their co-dominant inheritance. In the present study, we developed new SSR markers with next-generation sequencing using the Roche 454 GS FLX+ platform. Five hundred SSR primer sets were tested for the genetic identification of pineapple, including 100 each for the di-, tri-, tetra-, penta-, and hexa-nucleotide motif SSRs. In total, 160 SSR markers successfully amplified fragments and exhibited polymorphism among accessions. The SSR markers revealed the number of alleles per locus (ranging from 2 to 13), the expected heterozygosity (ranging from 0.041 to 0.823), and the observed heterozygosity (ranging from 0 to 0.875). A total of 117 SSR markers with tri- or greater nucleotide motifs were shown to be effective at facilitating accurate genotyping. Using the SSR markers, 25 accessions were distinguished genetically, with the exception of accessions 'MD-2' and 'Yonekura'. The developed SSR markers could facilitate the establishment of efficient and accurate genetic identification systems and the construction of genetic linkage maps in the future.

Identification of candidate genes responsible for the susceptibility of apple (Malus × domestica Borkh.) to Alternaria blotch.

BACKGROUND: The mechanism underlying the interaction between host plant and host-selective toxin (HST)-producing Alternaria alternata during infection is of particular interest for sustainable crop production. Alternaria blotch of apple (Malus × domestica Borkh.) caused by A. alternata apple pathotype is a major disease particularly in East Asia, which is the largest producer of apples globally. A single dominant gene, Alt, controls the susceptibility of the apple cultivar 'Delicious' to Alternaria blotch. In this study, we fine mapped the Alt locus and characterized three potential candidate genes. RESULTS: We used 797 F1 individuals derived from 15 crosses between apple accessions susceptible (Alt/alt) and resistant (alt/alt) to Alternaria blotch to construct physical and genetic maps of the Alt locus located on the top of chromosome 11. Susceptible accessions were derived from 'Delicious.' To fine map the Alt locus, we constructed a BAC library of 'Starking Delicious,' a sport of 'Delicious,' and used graphical genotyping to delimit the Alt locus to a region of 43 kb. Three genes predicted within the candidate Alt region were potentially involved in plant defense response, among which the gene encoding a coiled coil-nucleotide binding-leucine rich repeat (CC-NB-LRR) type disease resistance protein was the most promising. Moreover, a 12-bp insertion was uniquely identified in the 5' untranslated region of the Alt-associated allele of this gene, the presence or absence of which co-segregated with the susceptibility or resistance to A. alternata apple pathotype, respectively, among 43 tested cultivars including old ones and founders of modern apple breeding. CONCLUSION: A disease resistance protein has been suggested as a determinant of susceptibility/resistance to HST-producing A. alternata for the first time. Our finding provides new insight into the mechanism of HST-mediated disease control used by A. alternata against host plants.

The use of a fertile doubled haploid apple line for QTL analysis of fruit traits.

Apple is an economically important crop, and various approaches to genetic analysis in breeding programs have been attempted, including the production of doubled haploid (DH) lines, which are genetically homozygous. In this study, we used a DH line for QTL analyses, for the first time in a fruit tree, expecting it to simplify the analysis of the inheritance of quantitative traits and thus to enhance QTL detection power. Using an F1 population from 'Prima' × 'Apple Chukanbohon 95P6' (DH), we constructed a genetic map of 'Prima', and identified 19 QTLs for 13 traits. These QTLs had comparatively high LOD scores and explained a large part of the variation of the phenotypes. In particular, acidity, juice browning, and skin splitting clearly segregated at a 1:1 ratio, consistent with the segregation of the alleles at the detected QTLs in linkage group 16; these traits appeared to be regulated by single genes, despite general consideration that they are quantitative traits. Using this simple genetic composition of the F1 population, we concluded that the skin splitting of apple fruit has recessive inheritance, and that the allele for splitting is tightly linked with those for high acidity and low juice browning in 'Prima'.

Expression of a putative dioxygenase gene adjacent to an insertion mutation is involved in the short internodes of columnar apples (Malus × domestica).

Determining the molecular mechanism of fruit tree architecture is important for tree management and fruit production. An apple mutant 'McIntosh Wijcik', which was discovered as a bud mutation from 'McIntosh', exhibits a columnar growth phenotype that is controlled by a single dominant gene, Co. In this study, the mutation and the Co gene were analyzed. Fine mapping narrowed the Co region to a 101 kb region. Sequence analysis of the Co region and the original wild-type co region identified an insertion mutation of an 8202 bp long terminal repeat (LTR) retroposon in the Co region. Segregation analysis using a DNA marker based on the insertion polymorphism showed that the LTR retroposon was closely associated with the columnar growth phenotype. RNA-seq and RT-PCR analysis identified a promising Co candidate gene (91071-gene) within the Co region that is specifically expressed in 'McIntosh Wijcik' but not in 'McIntosh'. The 91071-gene was located approximately 16 kb downstream of the insertion mutation and is predicted to encode a 2-oxoglutarate-dependent dioxygenase involved in an unknown reaction. Overexpression of the 91071-gene in transgenic tobaccos and apples resulted in phenotypes with short internodes, like columnar apples. These data suggested that the 8202 bp retroposon insertion in 'McIntosh Wijcik' is associated with the short internodes of the columnar growth phenotype via upregulated expression of the adjacent 91071-gene. Furthermore, the DNA marker based on the insertion polymorphism could be useful for the marker-assisted selection of columnar apples.

Genomics of pear and other Rosaceae fruit trees.

The family Rosaceae includes many economically important fruit trees, such as pear, apple, peach, cherry, quince, apricot, plum, raspberry, and loquat. Over the past few years, whole-genome sequences have been released for Chinese pear, European pear, apple, peach, Japanese apricot, and strawberry. These sequences help us to conduct functional and comparative genomics studies and to develop new cultivars with desirable traits by marker-assisted selection in breeding programs. These genomics resources also allow identification of evolutionary relationships in Rosaceae, development of genome-wide SNP and SSR markers, and construction of reference genetic linkage maps, which are available through the Genome Database for the Rosaceae website. Here, we review the recent advances in genomics studies and their practical applications for Rosaceae fruit trees, particularly pear, apple, peach, and cherry.

Physical mapping of black spot disease resistance/susceptibility-related genome regions in Japanese pear (Pyrus pyrifolia) by BAC-FISH.

Black spot disease, caused by Alternaria alternata Japanese pear pathotype, is one of the most harmful diseases in Japanese pear cultivation. In the present study, the locations of black spot disease resistance/susceptibility-related genome regions were studied by fluorescence in situ hybridization using BAC clone (BAC-FISH) on Japanese pear (Pyrus pyrifolia (Burm. f.) Nakai) chromosomes. Root tips of self-pollinated seedlings of 'Osa Gold' were used as materials. Chromosome samples were prepared by the enzymatic maceration and air-drying method. The BAC clone adjacent to the black spot disease-related gene was labeled as a probe for FISH analysis. Black spot disease-related genome regions were detected in telomeric positions of two medium size chromosomes. These two sites and six telomeric 18S-5.8S-25S rDNA sites were located on different chromosomes as determined from the results of multi-color FISH. The effectiveness of the physical mapping of useful genes on pear chromosomes achieved by the BAC-FISH method was unequivocally demonstrated.

Fine mapping of the gene for susceptibility to black spot disease in Japanese pear (Pyrus pyrifolia Nakai).

Black spot disease, which is caused by the Japanese pear pathotype of the filamentous fungus Alternaria alternata (Fries) Keissler, is one of the most harmful diseases in Japanese pear cultivation. We mapped a gene for susceptibility to black spot disease in the Japanese pear (Pyrus pyrifolia Nakai) cultivar 'Kinchaku' (Aki gene) at the top of linkage group 11, similar to the positions of the susceptibility genes Ani in 'Osa Nijisseiki' and Ana in 'Nansui'. Using synteny-based marker enrichment, we developed novel apple SSR markers in the target region. We constructed a fine map of linkage group 11 of 'Kinchaku' and localized the Aki locus within a 1.5-cM genome region between SSR markers Mdo.chr11.28 and Mdo.chr11.34. Marker Mdo.chr11.30 co-segregated with Aki in all 621 F1 plantlets of a 'Housui' × 'Kinchaku' cross. The physical size of the Aki region, which includes three markers (Mdo.chr11.28, Mdo.chr11.30, and Mdo.chr11.34), was estimated to be 250 Kb in the 'Golden Delicious' apple genome and 107 Kb in the 'Dangshansuli' Chinese pear genome. Our results will help to identify the candidate gene for susceptibility to black spot disease in Japanese pear.

Genomic dissection of a 'Fuji' apple cultivar: re-sequencing, SNP marker development, definition of haplotypes, and QTL detection.

'Fuji' is one of the most popular and highly-produced apple cultivars worldwide, and has been frequently used in breeding programs. The development of genotypic markers for the preferable phenotypes of 'Fuji' is required. Here, we aimed to define the haplotypes of 'Fuji' and find associations between haplotypes and phenotypes of five traits (harvest day, fruit weight, acidity, degree of watercore, and flesh mealiness) by using 115 accessions related to 'Fuji'. Through the re-sequencing of 'Fuji' genome, total of 2,820,759 variants, including single nucleotide polymorphisms (SNPs) and insertions or deletions (indels) were detected between 'Fuji' and 'Golden Delicious' reference genome. We selected mapping-validated 1,014 SNPs, most of which were heterozygous in 'Fuji' and capable of distinguishing alleles inherited from the parents of 'Fuji' (i.e., 'Ralls Janet' and 'Delicious'). We used these SNPs to define the haplotypes of 'Fuji' and trace their inheritance in relatives, which were shown to have an average of 27% of 'Fuji' genome. Analysis of variance (ANOVA) based on 'Fuji' haplotypes identified one quantitative trait loci (QTL) each for harvest time, acidity, degree of watercore, and mealiness. A haplotype from 'Delicious' chr14 was considered to dominantly cause watercore, and one from 'Ralls Janet' chr1 was related to low-mealiness.

S-genotype identification based on allele-specific PCR in Japanese pear.

Gametophytic self-incompatibility in Japanese pear (Pyrus pyrifolia Nakai) is controlled by the single, multi-allelic S-locus. Information about the S-genotypes is important for breeding and the selection of pollen donors for fruit production. Rapid and reliable S-genotype identification system is necessary for efficient breeding of new cultivars in Japanese pear. We designed S allele-specific PCR primer pairs for ten previously reported S-RNase alleles (S (1)-S (9) and S (k)) as simple and reliable method. Specific nucleotide sequences were chosen to design the primers to amplify fragments of only the corresponding S alleles. The developed primer pairs were evaluated by using homozygous S-genotypes (S (1)/S (1)-S (9)/S (9) and S (4sm)/S (4sm)) and 14 major Japanese pear cultivars, and found that S allele-specific primer pairs can identify S-genotypes effectively. The S allele-specific primer pairs developed in this study will be useful for efficient S-genotyping and for marker-assisted selection in Japanese pear breeding programs.

Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.).

To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a 'piping-leaf-type' cultivar, 'Yugafu', and a 'spiny-tip-leaf-type' variety, 'Yonekura'. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the 'spiny-leaf type' as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding.

Identification of QTLs for fruit quality traits in Japanese apples: QTLs for early ripening are tightly related to preharvest fruit drop.

Many important apple (Malus × domestica Borkh.) fruit quality traits are regulated by multiple genes, and more information about quantitative trait loci (QTLs) for these traits is required for marker-assisted selection. In this study, we constructed genetic linkage maps of the Japanese apple cultivars 'Orin' and 'Akane' using F1 seedlings derived from a cross between these cultivars. The 'Orin' map consisted of 251 loci covering 17 linkage groups (LGs; total length 1095.3 cM), and the 'Akane' map consisted of 291 loci covering 18 LGs (total length 1098.2 cM). We performed QTL analysis for 16 important traits, and found that four QTLs related to harvest time explained about 70% of genetic variation, and these will be useful for marker-assisted selection. The QTL for early harvest time in LG15 was located very close to the QTL for preharvest fruit drop. The QTL for skin color depth was located around the position of MYB1 in LG9, which suggested that alleles harbored by 'Akane' are regulating red color depth with different degrees of effect. We also analyzed soluble solids and sugar component contents, and found that a QTL for soluble solids content in LG16 could be explained by the amount of sorbitol and fructose.

Identification of QTLs controlling harvest time and fruit skin color in Japanese pear (Pyrus pyrifolia Nakai).

Using an F1 population from a cross between Japanese pear (Pyrus pyrifolia Nakai) cultivars 'Akiakari' and 'Taihaku', we performed quantitative trait locus (QTL) analysis of seven fruit traits (harvest time, fruit skin color, flesh firmness, fruit weight, acid content, total soluble solids content, and preharvest fruit drop). The constructed simple sequence repeat-based genetic linkage map of 'Akiakari' consisted of 208 loci and spanned 799 cM; that of 'Taihaku' consisted of 275 loci and spanned 1039 cM. Out of significant QTLs, two QTLs for harvest time, one for fruit skin color, and one for flesh firmness were stably detected in two successive years. The QTLs for harvest time were located at the bottom of linkage group (LG) Tai3 (nearest marker: BGA35) and at the top of LG Tai15 (nearest markers: PPACS2 and MEST050), in good accordance with results of genome-wide association study. The PPACS2 gene, a member of the ACC synthase gene family, may control harvest time, preharvest fruit drop, and fruit storage potential. One major QTL associated with fruit skin color was identified at the top of LG 8. QTLs identified in this study would be useful for marker-assisted selection in Japanese pear breeding programs.

Genomic prediction of trait segregation in a progeny population: a case study of Japanese pear (Pyrus pyrifolia).

BACKGROUND: In cross breeding, it is important to choose a good parental combination that has high probability of generating offspring with desired characteristics. This study examines a method for predicting the segregation of target traits in a progeny population based on genome-wide markers and phenotype data of parental cultivars. RESULTS: The proposed method combines segregation simulation and Bayesian modeling for genomic selection. Marker segregation in a progeny population was simulated based on parental genotypes. Posterior marker effects sampled via Markov Chain Monte Carlo were used to predict the segregation pattern of target traits. The posterior distribution of the proportion of progenies that fulfill selection criteria was calculated and used for determining a promising cross and the necessary size of the progeny population. We applied the proposed method to Japanese pear (Pyrus pyrifolia Nakai) data to demonstrate the method and to show how it works in the selection of a promising cross. Verification using an actual breeding population suggests that the segregation of target traits can be predicted with reasonable accuracy, especially in a highly heritable trait. The uncertainty in predictions was reflected on the posterior distribution of the proportion of progenies that fulfill selection criteria. A simulation study based on the real marker data of Japanese pear cultivars also suggests the potential of the method. CONCLUSIONS: The proposed method is useful to provide objective and quantitative criteria for choosing a parental combination and the breeding population size.

Potential assessment of genome-wide association study and genomic selection in Japanese pear Pyrus pyrifolia.

Although the potential of marker-assisted selection (MAS) in fruit tree breeding has been reported, bi-parental QTL mapping before MAS has hindered the introduction of MAS to fruit tree breeding programs. Genome-wide association studies (GWAS) are an alternative to bi-parental QTL mapping in long-lived perennials. Selection based on genomic predictions of breeding values (genomic selection: GS) is another alternative for MAS. This study examined the potential of GWAS and GS in pear breeding with 76 Japanese pear cultivars to detect significant associations of 162 markers with nine agronomic traits. We applied multilocus Bayesian models accounting for ordinal categorical phenotypes for GWAS and GS model training. Significant associations were detected at harvest time, black spot resistance and the number of spurs and two of the associations were closely linked to known loci. Genome-wide predictions for GS were accurate at the highest level (0.75) in harvest time, at medium levels (0.38-0.61) in resistance to black spot, firmness of flesh, fruit shape in longitudinal section, fruit size, acid content and number of spurs and at low levels (<0.2) in all soluble solid content and vigor of tree. Results suggest the potential of GWAS and GS for use in future breeding programs in Japanese pear.

DNA profiling of pineapple cultivars in Japan discriminated by SSR markers.

We developed 18 polymorphic simple sequence repeat (SSR) markers in pineapple (Ananas comosus) by using genomic libraries enriched for GA and CA motifs. The markers were used to genotype 31 pineapple accessions, including seven cultivars and 11 breeding lines from Okinawa Prefecture, 12 foreign accessions and one from a related species. These SSR loci were highly polymorphic: the 31 accessions contained three to seven alleles per locus, with an average of 4.1. The values of expected heterozygosity ranged from 0.09 to 0.76, with an average of 0.52. All 31 accessions could be successfully differentiated by the 18 SSR markers, with the exception of 'N67-10' and 'Hawaiian Smooth Cayenne'. A single combination of three markers TsuAC004, TsuAC010 and TsuAC041, was enough to distinguish all accessions with one exception. A phenogram based on the SSR genotypes did not show any distinct groups, but it suggested that pineapples bred in Japan are genetically diversed. We reconfirmed the parentage of 14 pineapple accessions by comparing the SSR alleles at 17 SSR loci in each accession and its reported parents. The obtained information will contribute substantially to protecting plant breeders' rights.

Development of cultivar-specific DNA markers based on retrotransposon-based insertional polymorphism in Japanese pear.

We developed retrotransposon-based insertional polymorphism (RBIP) markers based on the long terminal repeat (LTR) sequences of copia-like retrotransposon Ppcrt4 and flanking genome sequences, which were derived from 454 sequencing data from Japanese pear (Pyrus pyrifolia) 'Hosui'. Out of 40 sequences including both LTR and flanking genome regions, we developed 22 RBIP markers and used them for DNA profiling of 80 pear cultivars: 64 Japanese, 10 Chinese (Pyrus ussuriensis) and 6 European (Pyrus communis). Three RBIP markers were enough to differentiate 'Hosui' from the other Japanese pear cultivars. The 22 RBIP markers could also distinguish 61 of the 64 Japanese pear cultivars. European pears showed almost no amplification of the 22 RBIP markers, which might suggest that retrotransposons had transposed during Asian pear evolution or reflect the genetic relationship between Asian and European pears. Sixteen of the RBIP markers could be positioned on a genetic linkage map of 'Hosui'. The RBIP loci were distributed in 10 linkage groups, and some loci were very closely located within the same linkage group. The information obtained will be applicable to developing cultivar-specific RBIP marker sets in plants.

Chromosome-level genome assembly of Japanese chestnut (Castanea crenata Sieb. et Zucc.) reveals conserved chromosomal segments in woody rosids.

Japanese chestnut (Castanea crenata Sieb. et Zucc.), unlike other Castanea species, is resistant to most diseases and wasps. However, genomic data of Japanese chestnut that could be used to determine its biotic stress resistance mechanisms have not been reported to date. In this study, we employed long-read sequencing and genetic mapping to generate genome sequences of Japanese chestnut at the chromosome level. Long reads (47.7 Gb; 71.6× genome coverage) were assembled into 781 contigs, with a total length of 721.2 Mb and a contig N50 length of 1.6 Mb. Genome sequences were anchored to the chestnut genetic map, comprising 14,973 single nucleotide polymorphisms (SNPs) and covering 1,807.8 cM map distance, to establish a chromosome-level genome assembly (683.8 Mb), with 69,980 potential protein-encoding genes and 425.5 Mb repetitive sequences. Furthermore, comparative genome structure analysis revealed that Japanese chestnut shares conserved chromosomal segments with woody plants, but not with herbaceous plants, of rosids. Overall, the genome sequence data of Japanese chestnut generated in this study is expected to enhance not only its genetics and genomics but also the evolutionary genomics of woody rosids.

Genetic structure analysis of cultivated and wild chestnut populations reveals gene flow from cultivars to natural stands.

Japanese chestnut (Castanea crenata Sieb. et Zucc.), the only fruit tree species domesticated in Japan, has been cultivated alongside natural stands since prehistorical times. Understanding the genetic diversity of this species and the relationships between cultivated and wild chestnut is important for clarifying its breeding history and determining conservation strategies. We assessed 3 chestnut cultivar populations and 29 wild chestnut populations (618 accessions). Genetic distance analysis revealed that wild populations in the Kyushu region are genetically distant from other populations, whereas other wild and cultivar populations are comparatively similar. Assignment tests suggested that cultivars were relatively similar to populations from central to western Honshu. Bayesian structure analyses showed that wild individuals were roughly classified according to geographical distribution along the Japanese archipelago, except that some wild individuals carried the genetic cluster prevalent in cultivars. Parentage analyses between cultivars and wild individuals identified 26 wild individuals presumed to have a parent-offspring relationship with a cultivar. These results suggested that the genetic structure of some wild individuals in natural stands was influenced by gene flow from cultivars. To conserve wild individuals carrying true "wild" genetic clusters, these individuals should be collected and preserved by ex situ conservation programs.