Integrated Reference Genetic Linkage Maps of Pear Based on SSR and AFLP Markers

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1 Breeding Science 57 : (2007) Integrated Reference Genetic Linkage Maps of Pear Based on SSR and AFLP Markers Toshiya Yamamoto* 1,2), Tetsuya Kimura 3), Shingo Terakami 1,2), Chikako Nishitani 1), Yutaka Sawamura 1), Toshihiro Saito 1), Kazuo Kotobuki 1) and Tateki Hayashi 1) 1) National Institute of Fruit Tree Science, 2-1 Fujimoto, Tsukuba, Ibaraki , Japan 2) Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba, Ibaraki , Japan 3) National Center for Seeds and Seedlings, 2-2 Fujimoto, Tsukuba, Ibaraki , Japan Integrated genetic linkage maps of the European pear (Pyrus communis L.) cultivars Bartlett and La France were constructed based on SSR markers developed from pear and apple, AFLP and other markers. The map of Bartlett, which was constructed using an F 1 population derived from a cross between Bartlett and Housui, consisted of 447 loci, including 58 loci revealed by pear SSR markers (hereafter referred to as pear SSR loci ), 60 by apple SSR markers (hereafter referred to as apple SSR loci ) and 322 by AFLP markers (hereafter referred to as AFLP loci ). This map covered 17 linkage groups over a total length of 1,000 cm with an average distance of 2.3 cm between markers. Another genetic linkage map of La France was constructed using F 1 individuals obtained from a cross between Shinsei and ( Housui La France ). The map of La France contained 414 loci, including 66 pear SSR loci, 68 apple SSR loci and 279 AFLP loci on 17 linkage groups encompassing a genetic distance of 1,156 cm. Using 97 common SSR markers, these two maps were well aligned together for all the 17 linkage groups, which corresponded to the basic chromosome number (n = 17). The positions of 66 SSR markers originating from apple were successfully determined in pear maps and showed a co-linearity with the saturated reference map of apple. Since the highdensity genetic linkage maps of pear constructed in the present study covered almost the entire genome, they should be considered as pear reference maps. These pear reference maps may enable to identify the location of genes of interest and QTLs (quantitative trait loci), and to analyze the syntenic genome structure with other Maloideae species. Key Words: AFLP, genetic map, Pyrus communis, simple sequence repeats. Introduction High-density genetic linkage maps are very useful for fundamental and applied genetic research. Linkage maps enable to conduct studies on the genome structure, the localization of genes of interest, the identification of quantitative trait loci (QTLs) and marker-assisted selection. Several genetic linkage maps of pears have been reported. Iketani et al. (2001) reported the construction of RAPD (random amplified polymorphic DNA)-based genetic maps of the Japanese pear varieties Kinchaku and Kousui. The map of Kinchaku consisted of 120 loci on 18 linkage groups (LGs) covering a length of ca. 770 cm. Partial genetic linkage maps of the European pear cultivars Passe Crassane, Harrow Sweet, Abbe Fetal and Max Red Bartlett were constructed using apple SSR (simple sequence repeat) markers, showing three LGs corresponding to the apple LGs 10, 12 and 14 (Pierantoni et al. 2004). Dondini et al. (2004) described two Communicated by M. Tahara Received May 11, Accepted September 1, *Corresponding author ( toshiya@affrc.go.jp) genetic linkage maps of the European pears Passe Crassane and Harrow Sweet. The map of Passe Crassane contained 155 loci for a total length of 912 cm organized on 18 LGs. These genetic linkage maps were, however, neither dense nor saturated. In our previous studies, genetic linkage maps of the European pear cultivar Bartlett and the Japanese pear cultivar Housui were constructed based on AFLP (amplified fragment length polymorphism) markers, SSR markers (from pear, apple and Prunus), isozymes, and phenotypic traits by using F 1 progenies (Yamamoto et al. 2002c, 2004). The map of the female parent Bartlett consisted of 256 loci, including 178 AFLP and 76 SSR (32 pear, 39 apple, 5 Prunus SSRs) loci, one isozyme and one self-incompatibility gene on 19 LGs over a total length of 1,020 cm (Yamamoto et al. 2004). However, the map contained 19 LGs, which did not match with the basic chromosome number (n = 17) of pear. In order to construct more accurate genetic maps of pear, it is necessary to analyze larger numbers of DNA markers covering entire genome regions. Pear (Pyrus spp.) belongs to the subfamily Maloideae in the family Rosaceae. This subfamily, characterized by a

2 322 Yamamoto, Kimura, Terakami, Nishitani, Sawamura, Saito, Kotobuki and Hayashi distinctive pome fruit, includes approximately 1,000 species in 30 genera (Westwood 1978), some of which are important fruit tree species, such as apple (Malus spp.), pear (Pyrus spp.), quince (Cydonia oblonga Mill.), loquat (Eriobotrya japonica (Thunb.) Mill.), medlar (Mespilus germanica L.), hawthorn (Crataegus spp.) and others (Kovanda 1965, Westwood 1978, Luby 2003). The basic chromosome number is n = 17 for all the Maloideae genera (Sax 1931, 1932, Kovanda 1965). In the subfamily Maloideae, two saturated (or highdensity) maps of apple have been published. The first one was produced by Maliepaard et al. (1998) based on the F 1 progeny of a cross between the apple cultivars Prima and Fiesta, using RFLP markers and isozymes. The maps of the parents were well aligned with 67 multi-allelic molecular markers, in which 17 LGs were identified, putatively corresponding to the basic chromosome number. The second map was based on 267 F 1 progenies from a cross between Fiesta and Discovery (Liebhard et al. 2002, 2003). The maps of Fiesta and Discovery, containing 115 and 112 SSR markers, respectively, could be integrated and anchored with ca. 100 SSR loci. Recently, a new set of ca. 150 SSR markers was mapped on the apple reference maps of Fiesta and Discovery (Silfverberg-Dilworth et al. 2006). Transferability of DNA markers within Maloideae and co-linearity between pear and apple have been investigated. SSR markers developed in apple produced discrete amplified fragments in several Pyrus genotypes, indicating that apple SSR markers could be applicable to pear (Yamamoto et al. 2001). Liebhard et al. (2002) reported that apple SSR markers successfully produced amplified fragments in species of other Maloideae genera (Amelanchier, Cotoneaster, Crataegus, Cydonia, Mespilus, Pyrus and Sorbus). In our previous study, LGs of Bartlett and Housui pears were well aligned with those of the apple Fiesta and Discovery reference maps by apple SSR markers (Yamamoto et al. 2004). These findings suggest that the positions and linkages of SSR loci might be well conserved between pear and apple. Pierantoni et al. (2004) also reported that more than 100 apple SSR markers were positioned on genetic linkage maps of pear, suggesting the existence of partial syntenic relationships between pear and apple. High-density or saturated genetic linkage maps of pear should reveal the existence of these syntenic relationships. In the present study, an integrated genetic linkage map was constructed for the European pear cultivar La France based on SSR markers developed from pear and apple, AFLP, and other markers. Our previous map for Bartlett (Yamamoto et al. 2002c, 2004) was further refined by mapping additional SSR and AFLP loci. Both maps consisted of more than 400 loci and showed LGs with the basic chromosome number of pear (n = 17). The two maps were well aligned, and their co-linearity and genome synteny with apple maps were examined. Materials and Methods Plant materials and DNA extraction Genetic linkage map of the European pear (Pyrus communis L.) cultivar La France was constructed using 55 F 1 individuals obtained from an interspecific cross between the Japanese pear Shinsei (Pyrus pyrifolia Nakai) and (the Japanese pear Housui La France ). The same set of 63 F 1 individuals from an interspecific cross between Bartlett and Housui described in our previous study (Yamamoto et al. 2002c, 2004), was used to refine the genetic linkage map of Bartlett. The double pseudo-testcross strategy (Grattapaglia and Sederoff 1994) was adopted to construct genetic linkage maps because of the selfincompatibility character of pear (Kikuchi 1929, Ishimizu et al. 1998). All the plant materials were obtained from the National Institute of Fruit Tree Science (Tsukuba, Ibaraki, Japan). Genomic DNA was isolated from young leaves by the CTAB-based extraction method (Hasebe and Iwatsuki 1990, Yamamoto et al. 2001). SSR analysis Ninety-nine SSR markers originating from pear were used to detect the microsatellite loci for La France (Yamamoto et al. 2002a, 2002b, 2002c, Sawamura et al. 2004, AB AB302443). For the Bartlett map, 54 out of the 99 pear SSR markers used for La France had been allocated previously (Yamamoto et al. 2002c, 2004). The other 45 markers were analyzed in the present study (Sawamura et al. 2004, AB AB302443). A total of 111 SSR markers developed from apple (Guilford et al. 1997, Gianfranceschi et al. 1998, Liebhard et al. 2002) were used for the genetic mapping of La France. Sixteen, 8 and 87 apple SSR markers used in the present study had been reported by Gianfranceschi et al. (1998), Guilford et al. (1997) and Liebhard et al. (2002), respectively. Although Liebhard et al. (2002) had developed 140 SSR markers, we selected 87 markers from those, whose linkage groups had already been identified. In the present study, 27 additional apple SSR markers (CH02a08, CH02a10, CH02c02a, CH03a02, CH03b06, CH03b10, CH03c02, CH03d08, CH03d10, CH03g04, CH04c06, CH04c07, CH04d07, CH04d10, CH04f06, CH04g09, CH04g10, CH05d03, CH05d11, CH05e03, CH05e05, CH05g07, CH05g11, COLa, MS01a03, MS02a01, MS06g03, Liebhard et al. 2002) were used to refine the genetic map of Bartlett. Overall, a total of 111 apple SSR markers were used for mapping. Microsatellite amplification was performed in a 20 µl reaction solution containing 10 mm Tris-HCl (ph 8.3), 50 mm KCl, 1.5 mm MgCl 2, 0.001% gelatin, 0.2 mm each of dntps, 10 pmoles of each forward primer labeled with fluorescent chemical (Fam/Tet/Hex, Fam/Vic/Ned) and unlabelled reverse primer, 10 ng of genomic DNA, and 0.5 unit of Taq polymerase (Invitrogen). Amplification was performed as follows: 35 cycles at 94 C for 1 min, C for

3 Reference genetic linkage map of pear min and 72 C for 2 min, for denaturation, annealing and primer extension, respectively. The PCR products were separated and detected using a PRISM 377 DNA sequencer or a Genetic Analyzer 3100 DNA sequencer (Applied Biosystems). The size of the amplified bands was determined based on an internal standard DNA (GeneScan-350TAMRA or 400HD-Rox, Applied Biosystems) using GeneScan software (Applied Biosystems). Analysis of AFLP and other markers AFLP analysis was performed using AFLP Analysis System II (Invitrogen), according to the supplier s protocol, except for the use of EcoRI primers labeled with fluorescent chemical. Two-hundred and fifty ng of genomic DNA was digested with two restriction enzymes (MseI, EcoRI) and then the DNA fragments were ligated to adaptors. Preamplification reactions were performed with a pre-amp primer mix. Selective amplification was performed with 40 primer combinations of eight MseI primers (M-CAA, M-CAC, M-CAG, M-CAT, M-CTA, M-CTC, M-CTG, M-CTT) and five Fam-labeled EcoRI primers (E-AC, E-TG, E-GC, E- GG, E-CG) for genetic mapping of La France. Twentyfour combinations of eight MseI primers (M-CAA, M-CAC, M-CAG, M-CAT, M-CTA, M-CTC, M-CTG, M-CTT) and three Fam-labeled EcoRI primers (E-GG, E-CG, E-CC) were used for Bartlett and the detected loci were combined with the AFLP loci identified in our previous studies (Yamamoto et al. 2002c, 2004). Overall, 64 combinations of the MseI and EcoRI primers were used in the AFLP analyses for Bartlett. The PCR products were separated and detected using a PRISM 377 DNA sequencer. The size of the amplified bands was determined based on an internal standard DNA (GeneScan-500ROX, Applied Biosystems) using the GeneScan software. Other molecular markers, SSR markers from peach and cherry and self-incompatibility locus (S locus) described in our previous studies (Yamamoto et al. 2002c, 2004) were integrated into the genetic maps of Bartlett and La France. An aspartate aminotransferase (AAT, E.C ) isozyme, AAT1 (Yamamoto et al. 2002c) was also included in the Bartlett map. Linkage analysis JoinMap ver. 2.0 (Stam and van Ooijen 1995) was used to construct the genetic linkage maps of Bartlett and La France. The Kosambi function was used to convert recombination units into genetic distances. The mapping analysis was conducted using a minimum LOD score of 5.0 for Bartlett, while a minimum LOD score of 4.0 was used in the mapping analysis to determine the LGs of La France. Markers showing distorted segregation determined by a chisquare test were included for the construction of the linkage maps. The number of linkage groups for Bartlett and La France was assigned based on that of an apple linkage group (Maliepaard et al. 1998) using common apple SSRs as anchor loci (Liebhard et al. 2002). Results and Discussion Marker analysis A total of 210 SSR markers from pear and apple (99 pear SSRs and 111 apple SSRs) were examined to segregate two mapping populations, F 1 progeny of Bartlett Housui and three-way F 1 progeny of Shinsei ( Housui La France ). Fifty-eight loci revealed by 55 pear SSR markers and 60 loci by 55 apple SSR markers showed heterozygous segregation for the alleles of Bartlett (Table 1). Out of the 58 pear SSR loci, three showed a distorted segregation at the 0.1% level. Twelve SSR loci originating from apple showed a distortion at the 0.1 5% level. In total, 118 SSR loci from pear and apple showing heterozygous segregation for alleles were used for the construction of the genetic linkage map of Bartlett. Segregation for the alleles derived from La France was examined in the progeny of Shinsei ( Housui La France ). Sixty-six loci revealed by 62 pear SSR markers generated segregating heterozygous alleles in La France (Table 2), seven of which showing a distortion at the 0.1 5% level. Sixty-eight loci revealed by 59 apple SSR markers showed heterozygous segregation for the alleles in La France (Table 2). Among them, 10 SSR loci showed a distorted segregation. A total of 134 loci revealed by 121 SSR markers that showed a segregation for heterozygous Table 1. Number and type of markers, genetic distance, marker density and gaps for genetic linkage map of Bartlett 1) Linkage groups Marker type Ba1 Ba2 Ba3 Ba4 Ba5 Ba6 Ba7 Ba8 Ba9 Ba10 Ba11 Ba12 Ba13 Ba14 Ba15 Ba16 Ba17 Total SSRs (pear) SSRs (apple) SSRs (Prunus) AFLPs Others Total Genetic distance (cm) Average distance between markers Largest gap (cm) ) Genetic map of Bartlett contains 256 loci (178 AFLP loci and 76 SSR loci from pear, apple and Prunus, one isozyme and one selfincompatibility gene) published in Yamamoto et al. (2002c, 2004).

4 324 Yamamoto, Kimura, Terakami, Nishitani, Sawamura, Saito, Kotobuki and Hayashi Table 2. Number and type of markers, genetic distance, marker density and gaps for genetic linkage map of La France Linkage groups Marker type La1 La2 La3 La4 La5 La6 La7 La8 La9 La10 La11 La12 La13 La14 La15 La16 La17 Total SSRs (pear) SSRs (apple) AFLPs Others 1 1 Total Genetic distance (cm) Average distance between markers Largest gap (cm) alleles were utilized for the construction of the genetic linkage map of La France. AFLP analysis performed in the F 1 progeny of Bartlett Housui resulted in the production of 338 segregated amplified fragments derived from Bartlett, by pooling the fragments detected by 24 AFLP primer combinations in the present study and those detected by 40 combinations in our previous studies. The number of polymorphic fragments per primer combination over 64 combinations ranged from 0 to 15 (E-TG/M-CTG) in Bartlett, with an average number of 5.3. About 6.2% (21/338) of the AFLP fragments showed a distorted segregation. AFLP analysis performed with 40 combinations in the F 1 progeny of Shinsei ( Housui La France ) generated 286 discrete polymorphic bands derived from La France. The number of polymorphic fragments per primer combination ranged from 0 (E-AC/M-CTA, E-GC/M-CTA, E-GC/M-CTG, E-TG/M- CAG, E-TG/M-CTA) to 16 (E-GG/M-CTC) in La France, with an average number of 7.2. Distorted segregations were observed in 40 out of 286 AFLP fragments (13.9%). The PCR-RFLP method enabled to identify the polymorphic self-incompatibility alleles (S genotypes) in La France as in the case of Bartlett (Yamamoto et al. 2002c). Genetic linkage maps in pear The linkage map of Bartlett consisted of 447 loci, including 58 pear SSR loci, 60 apple SSR loci, 5 Prunus SSR loci, 322 AFLP loci, one AAT1 isozyme and one selfincompatibility gene (Table 1 and Fig. 1). All the SSR loci were grouped into the 17 LGs, whereas 16 out of 338 AFLP loci were not assigned to any linkage group. Seventeen linkage groups were identified which covered 1,016 cm with an average distance of 2.3 cm between the markers (Table 1). The number of LGs corresponded to the basic chromosome number of 17. The length of the LGs ranged from 71.3 cm (LG Ba1) to 46.0 cm (LG Ba12). Gaps larger than 20 cm were not found for any of the 17 LGs. Medium gaps around 15 cm were identified for LGs Ba1 (15.7 cm), Ba14 (15.5 cm) and Ba15 (16.4 cm), whereas the largest gap within a linkage group was less than 10 cm for 10 LGs. Two LGs carried markers showing distorted segregation. It was found that the bottom region of LG Ba9 showed a slight distortion and that almost all the regions of LG Ba14 showed a considerable distortion. The linkage map of La France contained 414 loci, including 66 pear SSR loci, 68 apple SSR loci, 279 AFLP loci and one S locus, on the 17 LGs encompassing a genetic distance of 1,156 cm with an average distance of 2.8 cm between the markers (Table 2 and Fig. 1). All the SSR loci were grouped into the 17 LGs, whereas 7 out of the 286 AFLP loci were not assigned to any linkage group. The length of the LGs ranged from 95.7 cm (LG La15) to 55.2 cm (LG La7). The largest gap was found for LG La13 with a genetic distance of 23.5 cm. The largest gap within a linkage group was less than 20 cm for the other LGs. It was observed that 3 chromosomal regions carried markers showing a distorted segregation: namely the bottom region of LG La2, the central part of the bottom region of LG La5 and the central part of the bottom region of LG La10. All the 17 LGs of Bartlett and La France could be related to each other by 97 common SSR loci. The LGs 10 and 14 of the two maps were well aligned with 10 and 11 anchor loci, respectively (Fig. 1). The other 15 LGs were connected between Bartlett and La France by 1 to 7 common SSR loci. Additionally, the relative positions of the common SSR loci within a linkage group were well conserved between Bartlett and La France, except for very few cases, presumably due to the presence of multi-locus SSRs. These results indicate that the genome structure was conserved within European pears to a very large extent. Although a genetic linkage map of Housui was constructed using the same F 1 progeny of Bartlett Housui, three LGs or chromosomal regions were not identified because neither SSR nor AFLP markers of these regions showed segregating alleles (fragments) for Housui (data not shown). A complete genetic linkage map of Housui should become available, after the elucidation of the genome organization of these lacking or non-identified regions for Housui. Fifteen multi-locus SSRs were identified on two different LGs. Two loci revealed by the apple SSR marker CH03g12 were identified on LGs 1 and 3, in the genetic linkage maps of Bartlett, La France and apple. The SSR marker CH04c06 revealed two loci located on LGs 10 and 17, in these genetic linkage maps. Four SSR markers NB124b (LGs 2 & 5), CH04g09 (LGs 5 & 10), CH02a08 (LGs 5 & 10) and CH05g07 (LGs 12 & 14), revealed two different loci in the genetic maps of Bartlett and

5 Reference genetic linkage map of pear 325 Fig. 1. Genetic linkage maps of the European pears Bartlett and La France, and the apple Discovery (Liebhard et al. 2003). Linkage groups are designated as Ba1 to Ba17 for Bartlett, as La1 to La17 for La France and as D1 to D17 for Discovery. Genetic distances and loci are listed on the right and left sides of a linkage group bar, respectively. The designation of the AFLP marker is based on the primer combination and the sizes expressed as bp (Eco primer/mse primer-size). SSR loci from pear and apple are underlined and denoted by italics, respectively. SSR loci identified in the genetic linkage map of Fiesta are denoted within a map of Discovery, with roughly estimated positions in parentheses. Self-incompatibility locus is denoted by S on the Ba17 and La17 linkage groups. Asterisks indicate markers showing distorted segregations in a chi-square test. Distortions at 5%, 1% and 0.1% levels of significance are indicated as *, ** and ***, respectively.

6 326 Yamamoto, Kimura, Terakami, Nishitani, Sawamura, Saito, Kotobuki and Hayashi Fig. 1. (continued) La France. Two of the SSR markers, NB119b (LGs 3 & 14) and NB110a (LGs 9 & 17), revealed two loci in a map of Bartlett. The other seven SSR markers also revealed two loci mapped at different positions in a map of La France, i.e., CH02c02a (LGs 2 & 15), NB129a (LGs 2 & 15), CH05a02 (LGs 8 & 15), CH01h02 (LGs 9 & 17), CH05c06 (LGs 13 & 16), NB133a (LGs 13 & 16) and NB126a (LGs 17 & 17). These results suggest the presence of duplicated genomic regions in the pear genome. Evaluation of pear genetic linkage maps To determine whether our pear genetic maps covered the entire genome, we compared them with the saturated (or high-density) apple reference maps of Discovery and Fiesta (Liebhard et al. 2002, 2003) (Fig. 1). Since pear is taxonomically very close to apple, we could evaluate pear maps of Bartlett and La France by comparing them with apple maps using common SSR markers. A total of 66 SSR loci derived from apple were detected in both pear and apple maps, and at least a single common SSR locus was found on

7 Reference genetic linkage map of pear 327 Fig. 1. (continued) each LG (Fig. 1). Total genetic distances of the apple reference maps of Discovery and Fiesta were 1,450 and 1,140 cm, respectively, whereas those of Bartlett and La France were 1,016 and 1,156 cm, respectively. Since these maps were constructed using the same software, JoinMap, the value of the total length of our pear maps, to some extent, was lower than that of the apple reference maps. Pear LGs 1, 2, 4, 11, 14, and 15 were anchored to the apple reference maps of Discovery and Fiesta, with three, five, four, three, eight and five SSR loci, respectively. Length of these

8 328 Yamamoto, Kimura, Terakami, Nishitani, Sawamura, Saito, Kotobuki and Hayashi LGs was somewhat different between pear and apple. Additionally, the region of ca. 15 cm at the top of LG 10 appeared to be missing in our pear maps. However, the genome coverage estimated by common anchor loci was similar between them, indicating that the pear maps covered almost the entire genome. The two pear genetic maps constructed in the present study allocated more than 400 loci over the entire genome. The number of LGs determined by the maps corresponded to the basic chromosome number of pear. Therefore, these maps can be considered to be pear reference maps. Synteny between pear and apple The transferability of SSR markers between pear and apple as well as within the sub-family Maloideae had been investigated. It was reported that SSR markers developed in apple produced discrete amplified fragments in other Maloideae genera (Pyrus, Amelanchier, Cotoneaster, Crataegus, Cydonia, Mespilus and Sorbus) (Yamamoto et al. 2001, Liebhard et al. 2002). A partially syntenic relationship between pear and apple was suggested based on the transferability and positional conservation of SSR markers (Yamamoto et al. 2004, Pierantoni et al. 2004). In the present study, a total of 66 apple SSR loci were successfully identified in the pear maps and allocated to the same LGs as those in the apple maps. The pear genetic linkage maps constructed here revealed clear syntenic relationships between pear and apple. These pear maps could be utilized as reference for comparing the genome structure with that of other Maloideae genera along with the apple maps. Several interesting characteristics have been identified on the genetic linkage maps of pear and apple. The position of the self-incompatibility locus (S locus) was conserved between pear and apple mapped at the bottom of LG 17 (Maliepaard et al. 1998). The susceptibility to black spot disease (caused by Alternaria alternate Japanese pear pathotype) in pear was identified in the genetic linkage map of Osa Nijisseiki in the top region of LG 11 (Terakami et al. 2007). The major susceptibility gene of Alternaria blotch disease (caused by Alternaria alternate apple pathotype) in apple was mapped at the top of LG 11 (Dr. Akada-Fukasawa, personal communication), indicating that the positions of the susceptibility genes to Alternaria alternate might be conserved between pear and apple. The resistance gene Vnk of the Japanese pear Kinchaku to pear scab disease (caused by Venturia nashicola) was identified in the central region of LG 1 (Terakami et al. 2006). The Vf gene originating from Malus floribunda 821, which conferred resistance to apple scab disease (caused by V. inaequalis), was detected in the distal part of LG 1 (Maliepaard et al. 1998). In the case of these two scab resistance genes located on LG 1, the locus was different between pear and apple (Terakami et al. 2006). Several other scab resistance genes in apple, i.e., Vg, Vb, Vd, Vh2, Vh4, Vbj, Vh8, Vr2 and Vm, have been identified and mapped on 5 LGs (LG 1, LG 2, LG 10, LG12, LG17) (Bus et al. 2005, Calenge et al. 2004, Gygax et al. 2004, Hemmat et al. 2002, Patocchi et al. 2004, 2005, Tartarini et al. 2004). It is important to identify pear scab resistance genes from several Pyrus germplasms accessions and to compare their map positions with those of apple genes. Synteny relationships of functional genes could be further evaluated using the reference maps of pear and apple. Acknowledgments Contribution No of the National Institute of Fruit Tree Science. The authors thank Drs. H. Iketani, T. Hirabayashi, A. Sato, N. Takada and T. Imai for their valuable suggestions. We also thank Ms. T. Iida for her technical assistance. This research was partly supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Green Technology Project DM-1501). Literature Cited Bus, V.G.M., E.H.A. Rikkerink, W.E. van de Weg, R.L. Rusholme, S.E. Gardiner, H.C.M. Bassett, L.P. Kodde, L. Parisi, F.N.D. Laurens, E.J. Meulenbroek and K.M. Plummer (2005) The Vh2 and Vh4 scab resistance genes in two differential hosts derived from Russian apple R A map to the same linkage group of apple. Mol. Breed. 15: Calenge, F., A. Faure, M. Goerre, C. Gebhardt, W.E. Van de Weg, L.Parisi and C.E. Durel (2004) Quantitative trait loci (QTL) analysis reveals both broad-spectrum and isolate-specific QTL for scab resistance in an apple progeny challenged with eight isolates of Venturia inaequalis. Phytopathology 94: Dondini, L., L. Pierantoni, F. 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