Yutaka Sawamura

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.

Related polymorphic F-box protein genes between haplotypes clustering in the BAC contig sequences around the S-RNase of Japanese pear

Most fruit trees in the Rosaceae exhibit self-incompatibility, which is controlled by the pistil S gene, encoding a ribonuclease (S-RNase), and the pollen S gene at the S-locus. The pollen S in Prunus is an F-box protein gene (SLF/SFB) located near the S-RNase, but it has not been identified in Pyrus and Malus. In the Japanese pear, various F-box protein genes (PpSFBB-α–γ) linked to the S-RNase are proposed as the pollen S candidate. Two bacterial artificial chromosome (BAC) contigs around the S-RNase genes of Japanese pear were constructed, and 649 kb around S4-RNase and 378 kb around S2-RNase were sequenced. Six and 10 pollen-specific F-box protein genes (designated as PpSFBB4-u1–u4, 4-d1–d2 and PpSFBB2-u1–u5, 2-d1–d5, respectively) were found, but PpSFBB4-α–γ and PpSFBB2-γ were absent. The PpSFBB4 genes showed 66.2–93.1% amino acid identity with the PpSFBB2 genes, which indicated clustering of related polymorphic F-box protein genes between haplotypes near the S-RNase of the Japanese pear. Phylogenetic analysis classified 36 F-box protein genes of Pyrus and Malus into two major groups (I and II), and also generated gene pairs of PpSFBB genes and PpSFBB/Malus F-box protein genes. Group I consisted of gene pairs with 76.3–94.9% identity, while group II consisted of gene pairs with higher identities (>92%) than group I. This grouping suggests that less polymorphic PpSFBB genes in group II are non-S pollen genes and that the pollen S candidates are included in the group I PpSFBB genes.

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.

Dual recognition of S 1 and S 4 pistils by S 4 sm pollen in self-incompatibility of Japanese pear (Pyrus pyrifolia Nakai)

Most cultivars of Japanese pear (Pyrus pyrifolia Nakai) exhibit gametophytic self-incompatibility controlled by a single S-locus with multiple S-haplotypes. A self-compatible (SC) cultivar, ‘Osanijisseiki’ (S2S4sm), arising by a bud mutation of ‘Nijisseiki’ (S2S4), has a stylar-part mutant S4sm-haplotype, which lacks the pistil S4 gene, which is the S4-RNase gene. To efficiently breed SC cultivars, we selected ‘Nashi Chuukanbohon Nou 1 Gou’ (‘NCN1’) harboring homozygous S4sm from a self-progeny of Osanijisseiki and crossed it with ‘Okusankichi’ (S5S7), ‘Hakkou’ (S4S5), or ‘Ri-14’ (S1S2). Fruit set (%) was compared after self-pollination of the trees in the three progenies. All trees derived from the three progenies were predicted to be SC, except for the S4S4sm trees in the progeny of NCN1 × Hakkou. However, S1S4sm trees in the progeny of NCN1 × Ri-14 proved to be self-incompatible (SI). The pollen from Osanijisseiki was incompatible with ‘Doitsu’ (S1S2), but that from Nijisseiki was compatible, suggesting a possibility that the S4sm pollen was rejected by S1-harboring pistils. This possibility was clarified by crossing the pollen from NCN1 (S4smS4sm) to Doitsu, ‘Imamuraaki’ (S1S6), or ‘Hougetsu’ (S1S7), all of which proved incompatible. On the other hand, S4sm pollen was accepted by pistils harboring the S2, S3, S5, S6, S7, S9, and Sk haplotypes. The dual recognition of S1 and S4 pistils by S4sm pollen can be attributed to a mutation of the pollen S4 gene(s).

Mapping and pedigree analysis of the gene that controls the easy peel pellicle trait in Japanese chestnut (Castanea crenata Sieb. et Zucc.)

The Japanese chestnut (Castanea crenata Sieb. et Zucc.) has a pellicle that is difficult to peel, which increases the labor and cost for removing the pellicle from the nut during processing. Thus, a pellicle that is easier to peel has been an important objective of Japanese chestnut breeding programs. A newly released cultivar (“Porotan”) exhibits a unique, easily peeled pellicle. A previous study indicated that this trait is controlled by recessive gene p, and that several of the ancestors of Porotan (e.g., “Tanzawa” and 550-40) were P/p heterozygotes. Two F1 populations from intra-specific crosses of Japanese chestnut, Tanzawa (P/p) × Porotan (p/p) and 550-40 (P/p) × Tanzawa (P/p), were used for genetic mapping of the gene that controls this characteristic. A total of 11 simple sequence repeat (SSR) markers were obtained that showed significant linkages to the p gene, and genetic linkage maps for the region around the p gene were established. Pedigree analysis was conducted for eight ancestors of Porotan around the pellicle-peeling locus using graphical genotypes based on the 11 SSR loci. The two recessive p alleles and surrounding haplotypes of Porotan were inherited through different intermediate cultivars: one allele was derived from “Otomune” (P/p) via Tanzawa and the other was derived from Otomune via Tanzawa, “Kunimi” (P/p), and breeding line 550-40. A recombination event was found in the flanking region close to the p gene in Kunimi. Molecular identification of the easy peel pellicle trait will lead to marker-assisted selection and will greatly improve Japanese chestnut breeding.

Molecular characterization of novel Ty1-copia-like retrotransposons in pear (Pyrus pyrifolia)

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.

A segmental duplication encompassing S-haplotype triggers pollen-part self-compatibility in Japanese pear (Pyrus pyrifolia)

Self-compatible mutants of self-incompatible crops have been extensively studied for research and agricultural purposes. Until now, the only known pollen-part self-compatible mutants in Rosaceae subtribe Pyrinae, which contains many important fruit trees, were polyploid. This study revealed that the pollen-part self-compatibility of breeding selection 415-1, a recently discovered mutant of Japanese pear (Pyrus pyrifolia) derived from γ-irradiated pollen, is caused by a duplication of an S-haplotype. In the progeny of 415-1, some plants had three S-haplotypes, two of which were from the pollen parent. Thus, 415-1 was able to produce pollen with two S-haplotypes, even though it was found to be diploid: the relative nuclear DNA content measured by flow cytometry showed no significant difference from that of a diploid cultivar. Inheritance patterns of simple sequence repeat (SSR) alleles in the same linkage group as the S-locus (LG 17) showed that some SSRs closely linked to S-haplotypes were duplicated in progeny containing the duplicated S-haplotype. These results indicate that the pollen-part self-compatibility of 415-1 is not caused by a mutation of pollen S factors in either one of the S-haplotypes, but by a segmental duplication encompassing the S-haplotype. Consequently, 415-1 can produce S-heteroallelic pollen grains that are capable of breaking down self-incompatibility (SI) by competitive interaction between the two different S factors in the pollen grain. 415-1 is the first diploid pollen-part self-compatible mutant with a duplicated S-haplotype to be discovered in the Pyrinae. The fact that 415-1 is not polyploid makes it particularly valuable for further studies of SI mechanisms.