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.     

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.     

Construction of a high-density reference linkage map of tea (Camellia sinensis)

A few linkage maps of tea have been constructed using pseudo-testcross theory based on dominant marker systems. However, dominant markers are not suitable as landmark markers across a wide range of materials. Therefore, we developed co-dominant SSR markers from genomic DNA and ESTs and constructed a reference map using these co-dominant markers as landmarks. A population of 54 F₁ clones derived from reciprocal crosses between ‘Sayamakaori’ and ‘Kana-Ck17’ was used for the linkage analysis. Maps of both parents were constructed from the F₁ population that was taken for BC₁ population. The order of most of the dominant markers in the parental maps was consistent. We constructed a core map by merging the linkage data for markers that detected polymorphisms in both parents. The core map contains 15 linkage groups, which corresponds to the basic chromosome number of tea. The total length of the core map is 1218 cM. Here, we present the reference map as a central core map sandwiched between the parental maps for each linkage group; the combined maps contain 441 SSRs, 7 CAPS, 2 STS and 674 RAPDs. This newly constructed linkage map can be used as a basic reference linkage map of tea.     

Identificaiton of a clubroot resistance locus conferring resistance to a Plasmodiophora brassicae classified into pathotype group 3 in Chinese cabbage (Brassica rapa L.)

In Chinese cabbage (Brassica rapa), the clubroot resistance (CR) genes Crr1 and Crr2 are effective against the mild Plasmodiophora brassicae isolate Ano-01 and the more virulent isolate Wakayama-01, but not against isolate No. 14, classified into pathotype group 3. ‘Akiriso’, a clubroot-resistant F₁ cultivar, showed resistance to isolate No. 14. To increase the durability of resistance, we attempted to identify the CR locus in ‘Akiriso’. CR in ‘Akiriso’ segregated as a single dominant gene and was linked to several molecular markers that were also linked to CRb, a CR locus from cultivar ‘CR Shinki’. We developed additional markers around CRb and constructed partial genetic maps of this region in ‘Akiriso’ and ‘CR Shinki’. The positions and order of markers in the genetic maps of the two cultivars were very similar. The segregation ratios for resistance to isolate No. 14 in F₂ populations derived from each of the two cultivars were also very similar. These results suggest that the CR locus in ‘Akiriso’ is CRb or a tightly linked locus. The newly developed markers in this study were more closely linked to CRb than previously reported markers and will be useful for marker-assisted selection of CRb in Chinese cabbage breeding.     

Development of genomic and EST-SSR markers in radish (Raphanus sativus L.)

Radish (Raphanus sativus L.) belongs to Brassicaceae family and is a close relative of Brassica. This species shows a wide morphological diversity, and is an important vegetable especially in Asia. However, molecular research of radish is behind compared to that of Brassica. For example, reports on SSR (simple sequence repeat) markers are limited. Here, we designed 417 radish SSR markers from SSR-enriched genomic libraries and the cDNA data. Of the 256 SSR markers succeeded in PCR, 130 showed clear polymorphisms between two radish lines; a rat-tail radish and a Japanese cultivar, ‘Harufuku’. As a test case for evaluation of the present SSRs, we conducted two studies. First, we selected 16 SSRs to calculate polymorphism information contents (PICs) using 16 radish cultivars and four other Brassicaceae species. These markers detected 3–15 alleles (average = 9.6). PIC values ranged from 0.54 to 0.92 (average = 0.78). Second, part of the present SSRs were tested for mapping using our previously-examined mapping population. The map spanned 672.7 cM with nine linkage groups (LGs). The 21 radish SSR markers were distributed throughout the LGs. The SSR markers developed here would be informative and useful for genetic analysis in radish and its related species.     

Development and validation of DNA markers linked to Sdvy-1, a common bean gene conferring resistance to the yellowing strain of Soybean dwarf virus

The yellowing strain of Soybean dwarf virus (SbDV-YS) causes yellowing and yield loss in common bean (Phaseolus vulgaris). The most effective control is achieved through breeding for resistance. An indeterminate climbing cultivar with a white seed coat, ‘Oofuku’, is resistant to SbDV-YS in inoculation tests. We crossed ‘Oofuku’ with an elite cultivar, ‘Taisho-Kintoki’, which is SbDV-YS-susceptible, determinate dwarf with a red-purple seed coat, and performed amplified-fragment-length polymorphism analysis of F₃ lines. From nucleotide sequences of the resistant-specific fragments and their flanking regions, we developed five DNA markers, of which DV86, DV386, and DV398 were closely linked to Sdvy-1, a resistance gene. Using the markers, we developed ‘Toiku-B79’ and ‘Toiku-B80’, the near-isogenic lines (NILs) incorporating Sdvy-1 in the background of ‘Taisho-Kintoki’. The NILs had similar growth habit, maturity date and seed shape to those of ‘Taisho-Kintoki’. The quality of boiled beans was also similar, except that the NILs had more seed coat cracking than ‘Taisho-Kintoki’. The NILs showed no SbDV-YS infection in inoculation tests. We suggest that Sdvy-1 is a useful source of SbDV-YS resistance in common bean.     

Genome-wide indel markers shared by diverse Asian rice cultivars compared to Japanese rice cultivar ‘Koshihikari’

Insertion-deletion (indel) polymorphisms, such as simple sequence repeats, have been widely used as DNA markers to identify QTLs and genes and to facilitate rice breeding. Recently, next-generation sequencing has produced deep sequences that allow genome-wide detection of indels. These polymorphisms can potentially be used to develop high-accuracy polymerase chain reaction (PCR)-based markers. Here, re-sequencing of 5 indica, 2 aus, and 3 tropical japonica cultivars and Japanese elite cultivar ‘Koshihikari’ was performed to extract regions containing large indels (10–51 bp) shared by diverse cultivars. To design indel markers for the discrimination of genomic regions between ‘Koshihikari’ and other diverse cultivars, we subtracted the indel regions detected in ‘Koshihikari’ from those shared in other cultivars. Two sets of indel markers, KNJ8-indel (shared in eight or more cultivars, including ‘Khao Nam Jen’ as a representative tropical japonica cultivar) and C5-indel (shared in five to eight cultivars), were established, with 915 and 9,899 indel regions, respectively. Validation of the two marker sets by using 23 diverse cultivars showed a high PCR success rate (≥95%) for 83.3% of the KNJ8-indel markers and 73.9% of the C5-indel markers. The marker sets will therefore be useful for the effective breeding of Japanese rice cultivars.     

Evaluation of the effects of five QTL regions on Fusarium head blight resistance and agronomic traits in spring wheat (Triticum aestivum L.)

Fusarium head blight (FHB) is an important disease of wheat (Triticum aestivum L.). The aim of this study was to determine the effects of quantitative trait locus (QTL) regions for resistance to FHB and estimate their effects on reducing FHB damage to wheat in Hokkaido, northern Japan. We examined 233 F₁-derived doubled-haploid (DH) lines from a cross between ‘Kukeiharu 14’ and ‘Sumai 3’ to determine their reaction to FHB during two seasons under field conditions. The DH lines were genotyped at five known FHB-resistance QTL regions (on chromosomes 3BS, 5AS, 6BS, 2DL and 4BS) by using SSR markers. ‘Sumai 3’ alleles at the QTLs at 3BS and 5AS effectively reduced FHB damage in the environment of Hokkaido, indicating that these QTLs will be useful for breeding spring wheat cultivars suitable for Hokkaido. Some of the QTL regions influenced agronomic traits: ‘Sumai 3’ alleles at the 4BS and 5AS QTLs significantly increased stem length and spike length, that at the 2DL QTL significantly decreased grain weight, and that at the 6BS QTL significantly delayed heading, indicating pleiotropic or linkage effects between these agronomic traits and FHB resistance.     

Close linkage of a blast resistance gene,Pias(t), with a bacterial leaf blight resistance gene,Xa1-as(t), in a rice cultivar ‘Asominori’

It has long been known that a bacterial leaf blight-resistant line in rice obtained from a crossing using ‘Asominori’ as a resistant parent also has resistance to blast, but a blast resistance gene in ‘Asominori’ has not been investigated in detail. In the present study, a blast resistance gene in ‘Asominori’, tentatively named Pias(t), was revealed to be located within 162-kb region between DNA markers YX4-3 and NX4-1 on chromosome 4 and to be linked with an ‘Asominori’ allele of the bacterial leaf blight resistance gene Xa1, tentatively named Xa1-as(t). An ‘Asominori’ allele of Pias(t) was found to be dominant and difference of disease severity between lines having the ‘Asominori’ allele of Pias(t) and those without it was 1.2 in disease index from 0 to 10. Pias(t) was also closely linked with the Ph gene controlling phenol reaction, suggesting the possibility of successful selection of blast resistance using the phenol reaction. Since blast-resistant commercial cultivars have been developed using ‘Asominori’ as a parent, Pias(t) is considered to be a useful gene in rice breeding for blast resistance.     

Transfer of the Rsv3 locus from ‘Harosoy’ for resistance to soybean mosaic virus strains C and D in Japan

Resistance to soybean mosaic virus (SMV) is imperative for soybean (Glycine max (L.) Merr.) production in the Tohoku region. Molecular markers for SMV resistance were previously reported for U.S. SMV strains, but they cannot be applied because of the differences in strain classification between Japan and the U.S. A U.S. variety ‘Harosoy’ has been used mainly as a donor of resistance to SMV strains C and D in a Japanese breeding program, resulting in resistant varieties such as ‘Fukuibuki.’ Because ‘Harosoy’ harbors the Rsv3 gene conferring resistance to the virulent SMV strain groups, G5 through G7, it appears that the Rsv3 gene confers resistance to strains C and D. In this study, we introduced resistance to the two strains from ‘Fukuibuki’ into a leading variety ‘Ohsuzu’ by recurrent backcrossing with marker-assisted selection. All lines selected with markers near Rsv3 showed resistance to the strains, suggesting that the Rsv3 locus is responsible for the resistance. Three years of trials showed that one of the breeding lines, ‘Tohoku 169,’ was equivalent to ‘Ohsuzu’ with respect to agricultural characteristics such as seed size, maturity date, and seed yield, except for the SMV resistance.     

Parentage analysis of tea cultivars in Japan based on simple sequence repeat markers

Tea cultivars have been bred by individual selection of landraces and by crossbreeding, but the validation of the parentage is limited. In this study, we performed parentage analysis of 79 tea cultivars in Japan based on SSR markers to confirm or identify the parent-offspring relationships among them. The effectiveness of nine SSR markers for parentage analysis was validated by comparing them to the existing cleaved amplified polymorphic sequence markers. The former markers were detectable more alleles than the latter. Simulation of parentage analysis of the tea cultivars predicted biparental origins for 12 cultivars (‘Houshun’, ‘Mie ryokuhou no. 1’, ‘Surugawase’, ‘Tenmyo’, ‘Yamanoibuki’, ‘Harumidori’, ‘Koushun’, ‘Minekaori’, ‘Okumusashi’, ‘Saemidori’, ‘Sofu’, and ‘Toyoka’), in the first five of which candidate parents of yet-to-be-defined pedigree were newly identified. Comparisons of a total of 41 SSR genotypes confirmed the newly-identified parentages of ‘Asahi’ for ‘Tenmyo’, ‘Rokurou’ for ‘Houshun’, ‘Surugawase’, and ‘Yamanoibuki’, and ‘Yamatomidori’ for ‘Mie ryokuhou no. 1’. The maternity of seven cultivars out of the 12 was also confirmed with chloroplast DNA sequences. Uniparental origins were confirmed for 25 cultivars. This parentage analysis has improved our knowledge of tea pedigrees and will aid in the development of new cultivars.     

Genetic dissection of black grain rice by the development of a near isogenic line

Rice (Oryza sativa L.) can produce black grains as well as white. In black rice, the pericarp of the grain accumulates anthocyanin, which has antioxidant activity and is beneficial to human health. We developed a black rice introgression line in the genetic background of Oryza sativa L. ‘Koshihikari’, which is a leading variety in Japan. We used Oryza sativa L. ‘Hong Xie Nuo’ as the donor parent and backcrossed with ‘Koshihikari’ four times, resulting in a near isogenic line (NIL) for black grains. A whole genome survey of the introgression line using DNA markers suggested that three regions, on chromosomes 1, 3 and 4 are associated with black pigmentation. The locus on chromosome 3 has not been identified previously. A mapping analysis with 546 F₂ plants derived from a cross between the black rice NIL and ‘Koshihikari’ was evaluated. The results indicated that all three loci are essential for black pigmentation. We named these loci Kala1, Kala3 and Kala4. The black rice NIL was evaluated for eating quality and general agronomic traits. The eating quality was greatly superior to that of ‘Okunomurasaki’, an existing black rice variety. The isogenicity of the black rice NIL to ‘Koshihikari’ was very high.     

Major QTLs associated with green stem disorder insensitivity of soybean (Glycine max (L.) Merr.)

Green stem disorder (GSD) is one of the most serious syndromes affecting soybean (Glycine max) cultivation in Japan. In GSD, stems remain green even when pods mature. When soybean plants develop GSD, seed surfaces are soiled by tissue fluid and seed quality is deteriorated during machine harvesting. We performed quantitative trait locus (QTL) analyses for GSD insensitivity using recombinant inbred lines (RILs; n = 154) derived from a cross between an insensitive line (‘Touhoku 129’) and a sensitive leading cultivar (‘Tachinagaha’) during a 6-year evaluation. Three effective QTLs were detected. The influences of these QTLs were in the following order: qGSD1 (LG_H) > qGSD2 (LG_F) > qGSD3 (LG_L). At these three QTLs, ‘Touhoku 129’ genotypes exhibited more GSD insensitivity than ‘Tachinagaha’ genotypes. The lower incidence of GSD for ‘Touhoku129’ was attributable primarily to these three QTLs because RILs harboring a ‘Touhoku 129’ genotype at the three QTLs exhibited a GSD incidence similar to that of ‘Touhoku 129.’ Although a limitation of this study is that only one mapping population was evaluated, this QTL information and the flanking markers of these QTLs would be effective tools for resolving GSD in soybean breeding programs.     

Identification of quantitative trait loci for powdery mildew resistance in highly resistant cucumber (Cucumis sativus L.) using ddRAD-seq analysis

Powdery mildew, caused by Podosphaera xanthii (syn. Sphaerotheca fuliginea ex Fr. Poll.), is one of the most economically important foliar diseases in cucumber (Cucumis sativus L.). Cucumber parental line ‘Kyuri Chukanbohon Nou 5 Go’, developed from weedy cucumber line CS-PMR1, is highly resistant to powdery mildew and is promising breeding material. We performed quantitative trait locus (QTL) analysis using double-digest restriction-site-associated DNA sequencing (ddRAD-Seq) in a population from a cross between ‘Kyuri Chukanbohon Nou 5 Go’ and the Japanese native cultivar ‘Kaga-aonaga-fushinari’, which is susceptible to powdery mildew. The resistance of the population and its parents was evaluated using leaf disc assays and image analysis. We detected one major QTL on Chr. 5 that was effective at both 20°C and 25°C and one minor QTL on Chr. 1 effective at 20°C. We detected two additional QTLs in subpopulation: one on Chr. 3 effective at 20°C and one on Chr. 5 effective at both 20°C and 25°C in a position different from the major QTL. The resistance alleles at all four QTLs were contributed by ‘Kyuri Chukanbohon Nou 5 Go’. The results of this study can be used to develop practical DNA markers tightly linked to genes for powdery mildew resistance.     

Reverted glutathione S-transferase-like genes that influence flower color intensity of carnation (Dianthus caryophyllus L.) originated from excision of a transposable element

A glutathione S-transferase-like gene, DcGSTF2, is responsible for carnation (Dianthus caryophyllus L.) flower color intensity. Two defective genes, DcGSTF2mu with a nonsense mutation and DcGSTF2-dTac1 containing a transposable element dTac1, have been characterized in detail in this report. dTac1 is an active element that produces reverted functional genes by excision of the element. A pale-pink cultivar ‘Daisy’ carries both defective genes, whereas a spontaneous deep-colored mutant ‘Daisy-VPR’ lost the element from DcGSTF2-dTac1. This finding confirmed that dTac1 is active and that the resulting reverted gene, DcGSTF2rev1, missing the element is responsible for this color change. Crosses between the pale-colored cultivar ‘06-LA’ and a deep-colored cultivar ‘Spectrum’ produced segregating progeny. Only the deep-colored progeny had DcGSTF2rev2 derived from the ‘Spectrum’ parent, whereas progeny with pale-colored flowers had defective forms from both parents, DcGSTF2mu and DcGSTF2-dTac1. Thus, DcGSTF2rev2 had functional activity and likely originated from excision of dTac1 since there was a footprint sequence at the vacated site of the dTac1 insertion. Characterizing the DcGSTF2 genes in several cultivars revealed that the two functional genes, DcGSTF2rev1 and DcGSTF2rev2, have been used for some time in carnation breeding with the latter in use for more than half a century.     

Development of EST-SSR markers and construction of a linkage map in faba bean (Vicia faba)

To develop a high density linkage map in faba bean, a total of 1,363 FBES (Faba bean expressed sequence tag [EST]-derived simple sequence repeat [SSR]) markers were designed based on 5,090 non-redundant ESTs developed in this study. A total of 109 plants of a ‘Nubaria 2’ × ‘Misr 3’ F₂ mapping population were used for map construction. Because the parents were not pure homozygous lines, the 109 F₂ plants were divided into three subpopulations according to the original F₁ plants. Linkage groups (LGs) generated in each subpopulation were integrated by commonly mapped markers. The integrated ‘Nubaria 2’ × ‘Misr 3’ map consisted of six LGs, representing a total length of 684.7 cM, with 552 loci. Of the mapped loci, 47% were generated from multi-loci diagnostic (MLD) markers. Alignment of homologous sequence pairs along each linkage group revealed obvious syntenic relationships between LGs in faba bean and the genomes of two model legumes, Lotus japonicus and Medicago truncatula. In a polymorphic analysis with ten Egyptian faba bean varieties, 78.9% (384/487) of the FBES markers showed polymorphisms. Along with the EST-SSR markers, the dense map developed in this study is expected to accelerate marker assisted breeding in faba bean.     

Development of PCR-based DNA marker for detection of white carrot contamination caused by Y2 locus

In carrot (Daucus carota L.), the taproot colors orange, yellow and white are determined mostly by the Y, Y2, and Or loci. One of the most severe issues in carrot seed production is contamination by wild white carrot. To evaluate the contamination ratio, easily detectable DNA markers for white carrot are desired. To develop PCR-based DNA markers for the Y2 locus, we have re-sequenced two orange-colored carrot cultivars at our company (Fujii Seed, Japan), as well as six white- and one light-orange-colored carrots that contaminated our seed products. Within the candidate region previously reported for the Y2 locus, only one DNA marker, Y2_7, clearly distinguished white carrots from orange ones in the re-sequenced samples. The Y2_7 marker was further examined in 12 of the most popular hybrid orange cultivars in Japan, as well as ‘Nantes’ and ‘Chantenay Red Cored 2’. The Y2_7 marker showed that all of the orange cultivars examined had the orange allele except for ‘Beta-441’. False white was detected in the orange-colored ‘Beta-441’. The Y2_7 marker detected white root carrot contamination in an old open-pollinated Japanese cultivar, ‘Nakamura Senkou Futo’. This marker would be a useful tool in a carrot seed quality control for some cultivars.     

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 quantitative trait loci associated with boiled seed hardness in soybean

Boiled seed hardness is an important factor in the processing of soybean food products such as nimame and natto. Little information is available on the genetic basis for boiled seed hardness, despite the wide variation in this trait. DNA markers linked to the gene controlling this trait should be useful in soybean breeding programs because of the difficulty of its evaluation. In this report, quantitative trait locus (QTL) analysis was performed to reveal the genetic factors associated with boiled seed hardness using a recombinant inbred line population developed from a cross between two Japanese cultivars, ‘Natto-shoryu’ and ‘Hyoukei-kuro 3’, which differ largely in boiled seed hardness, which in ‘Natto-shoryu’ is about twice that of ‘Hyoukei-kuro 3’. Two significantly stable QTLs, qHbs3-1 and qHbs6-1, were identified on chromosomes 3 and 6, for which the ‘Hyoukei-kuro 3’ alleles contribute to decrease boiled seed hardness for both QTLs. qHbs3-1 also showed significant effects in progeny of a residual heterozygous line and in a different segregating population. Given its substantial effect on boiled seed hardness, SSR markers closely linked to qHbs3-1, such as BARCSOYSSR_03_0165 and BARCSOYSSR_03_0185, could be useful for marker-assisted selection in soybean breeding.     

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.