Sogo Nishio

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.

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.

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.

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 (S1–S9 and Sk) 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 (S1/S1–S9/S9 and S4sm/S4sm) 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.

Identification of QTLs Associated with Conversion of Sucrose to Hexose in Mature Fruit of Japanese Pear

Sweetness is the most important trait for fruit breeding and is fundamentally determined by both total and individual sugar contents. We analyzed the contents of sucrose, fructose, glucose, and sorbitol in mature fruit in an F1 population derived from crossing modern Japanese pear cultivar ‘Akizuki’ and breeding line ‘373-55’. A genetic linkage map was constructed using simple sequence repeats (SSRs) and single-nucleotide polymorphisms (SNP). We identified two regions associated with individual sugar contents on linkage group (LG) 1 and LG 7. The percentages of the variance in sucrose, fructose, and glucose explained by the quantitative trait loci (QTLs) were 26.6, 15.9, and 18.5%, respectively, for the region on LG 1, and 22.2, 20.0, and 9.5%, respectively, for the region on LG 7. In both regions, genotypes associated with increases in sucrose were associated with decreases in both fructose and glucose. The 1.5-LOD support intervals of the QTLs on LGs 1 and 7 include SSRs within the regions flanking acid invertase genes PPAIV3 and PPAIV1, respectively. Because acid invertase is a key enzyme in the conversion of sucrose to hexose, these are promising candidates for genes underlying those QTLs and controlling individual sugar contents. We also found one region on LG 11 that explained 21.4% of the variation in total sugar content but was not significantly associated with variation for individual sugars. The information obtained in this study will accelerate research and breeding programs to improve fruit sweetness.

Bayesian genome-wide association study of nut traits in Japanese chestnut

Japanese chestnut (Castanea crenata Sieb. et Zucc.) has a long juvenile phase, so breeders have to wait many years to evaluate nut traits. Molecular markers associated with genes of interest would accelerate selection in chestnut breeding programs. We evaluated five nut traits (nut harvest date, nut weight, pericarp splitting, insect infestation, and specific gravity) in 99 Japanese chestnut cultivars and selections. A wide range of phenotypic variation was observed for each of the traits, suggesting that the collection harbored sufficient genetic diversity for breeding. A Bayesian genome-wide association study was conducted using 162 simple sequence repeat markers and 741 single nucleotide polymorphism markers. To evaluate the potential of marker-assisted selection, we examined 12 molecular markers found to be associated with nut traits: 4 for nut harvest date, 4 for nut weight, 1 for pericarp splitting, and 3 for insect infestation. The percentages of phenotypic variance explained ranged from 4.8 to 37.1%. Although insect infestation showed only medium heritability (0.672), we obtained two quantitative trait loci (QTLs) with extremely high posterior probabilities (0.93 and 1.00). Out of the 12 molecular markers, 3 of the 4 markers for nut harvest time could be validated in a breeding population. Accuracies of genomic selection were extremely high for nut harvest date (0.841) and moderate for insect infestation (0.604), suggesting that genomic selection based on Bayesian regression would reduce the cost of phenotypic evaluation of these traits and possibly others.