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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.

Retrotransposons are present in all plant genomes and play important roles in genome size, genome structure remodeling, gene function, and genome evolution. Eight novel long terminal repeat retrotransposons were identified from a bacterial artificial chromosome library of Japanese pear (Pyrus pyrifolia). On the basis of the order of gene arrangement within the gag and pol domains (protease, integrase, reverse transcriptase, and RNase H), these newly identified retrotransposons appear to be closely related to Ty1-copia retrotransposons. They were designated Ppcrt1–8 and classified into two groups based on the presence or absence of a 142-amino-acid deletion within the group-specific antigen DNA-binding domain. Ppcrt1–8 were grouped with the copia-like retrotransposons RIRE1 and BARE-1 by phylogenetic analysis based on the amino acid sequences encoded by the gag and pol domains. Fluorescence in situ hybridization analysis showed that sequences homologous to Ppcrt4 were dispersed throughout more than half of the pear chromosomes. Southern blot analysis suggested that many copies of Ppcrt retrotransposons exist in the pear genome. Sequence information from these eight retrotransposons should be useful for the development of retrotransposon-based molecular marker systems in Japanese pear.

ADDITIONAL INDEX WORDS. androgenesis, doubled haploids, flow cytometry, Malus ·domestica, morphology, SSR analysis, S-allele ABSTRACT. Doubled haploids can improve the efficiency of breeding and genetic study in apple (Malus ·domestica Borkh.). Seventeen homozygous genotypes were obtained by in vitro anther culture from 'Senshu' apple. Flow cytometry analysis revealed that the ploidy level of the anther-derived plantlets was diploid. Simple sequence repeat (SSR) analysis determined the origin and homozygous status of the anther-derived plantlets. The results of S-RNase polymerase chain reaction (PCR)-digestion analysis reinforced the homozygous state. The morphological charac- teristics and reproductive potential of the doubled haploids were investigated. It is especially interesting that one of the doubled haploids derived from anther culture had fertile pollen grains (rate of in vitro germination was 20.6%- 33.0%), and several progenies were obtained from the cross between 'Prima' apple and the doubled haploid. The hybridism of these seedlings was confirmed by SSR analysis. Furthermore, the female gametes of the doubled haploid showed slight fertility. Thus, this doubled haploid will be valuable for breeding and genetic studies.

Information about self-incompatibility (S) genotypes of apple cultivars is important for the selection of pollen donors for fruit production and breeding. Although S genotyping systems using S haplotype-specific PCR of S-RNase, the pistil S gene, are useful, they are sometimes associated with false-positive/negative problems and are unable to identify new S haplotypes. The CAPS (cleaved amplified polymorphic sequences) system is expected to overcome these problems, however, the genomic sequences needed to establish this system are not available for many S-RNases. Here, we determined partial genomic sequences of eight S-RNases, and used the information to design new primer and to select 17 restriction enzymes for the discrimination of 22 S-RNases by CAPS. Using the system, the S genotypes of three cultivars were determined. The genomic sequence-based CAPS system would be useful for S genotyping and analyzing new S haplotypes of apple.

Apple exhibits gametophytic self-incompatibility (GSI) that is controlled by the multiallelic S-locus. This S-locus encodes polymorphicS ribonuclease (S-RNase) for the pistil-part 5 determinant. Information aboutS-genotypes is important when selecting pollen donors for fruit production and breeding of new cultivars. We determined the 5-genotypes of ‘Charden’ (S2S3S4), ‘Winesap’ (S1S28), ‘York Imperial’ (S2S31), ‘Stark Earliblaze’ (S1S28), and ‘Burgundy’ (S20S32), byS-RNase sequencing and S-allele-specific PCR analysis. Two newS-RNases, S31 and S32, were also identified from ‘York Imperial’ and ‘Burgundy’, respectively. These newS-alleles contained the conserved eight cysteine residues and two histidine residues essential for RNase activity. Whereas S31 showed high similarity to S20 (94%), S32 exhibited 58% (to S24) to 76% (to S25) similarity in the exon regions. We designed newS-allele-specific primers for amplifying S31- and S32-RNasc-specific fragments; these can serve as specific gene markers. We also rearranged the apple S-allele numbers containing those newS-RNases. They should be useful, along with anS-RNase-based PCR system, in determining S-genotypes and analyzing new alleles from apple cultivars.

Japanese pear exhibits gametophytic self-incompatibility that is controlled by the multiallelic S-locus. The S-locus encodes polymorphic S-RNase for the pistil-part S determinant in Japanese pear. Information about the S-genotypes is important for the selection of pollen donors for fruit production and breeding of Japanese pear cultivars. We determined the S-genotypes of three Japanese pear cultivars, ‘Kinchaku’ (S4Sk), ‘Natsuhikari’ (S3S4) and ‘Wakahikari’ (S3S4), and an F1 hybrid cultivar between a Japanese pear cultivar and a European pear cultivar, ‘Oharabeni’ (S9Se), by PCR-RFLP analysis of S-RNase. A new Sk-RNase allele was identified in ‘Kinchaku’, and full length cDNA encoding Sk-RNase was sequenced. The Sk-RNase was not effectively amplified by the previously published primers. We designed a new PCR primer combination for the amplification of the Sk-RNase allele in addition to the S1- to S9-RNase alleles of Japanese pear, and developed a PCR-RFLP system for identifying the S1- to S9- and Sk-alleles.

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.