Noriyuki Onoue

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.

CRISPR/Cas9-mediated targeted mutagenesis in grape

RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape—an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.

SSR-based molecular profiling of 237 persimmon ( Diospyros kaki Thunb.) germplasms using an ASTRINGENCY -linked marker

Pollination-constant non-astringent (PCNA) trait is desirable in persimmon production because it confers natural astringency loss in mature persimmon fruit. Expression of the PCNA trait requires six homozygous recessive PCNA (ast) alleles at the single ASTRINGENCY (AST) locus in hexaploid persimmon. When crossing non-PCNA accessions to breed PCNA offspring, knowledge of ast and non-PCNA (AST) allele dosage in the parental accessions is important, because more PCNA offspring can segregate from a non-PCNA parent with more ast and fewer AST alleles. Previously, we have demonstrated that a region linked to the AST locus has numerous fragment size polymorphisms with varying numbers of simple sequence repeats. Here, we reveal the polymorphisms in this region in a broad collection of persimmon germplasms. Among 237 accessions, we distinguished 21 AST- and 5 ast-linked fragments with different sizes. Based on the number of fragments detected per individual, we identified 21 non-PCNA accessions with three different ast alleles; by crossing these with a PCNA parent, we obtain PCNA offspring under autohexaploid inheritance. Furthermore, AST and ast allelic combination patterns in hexaploid persimmon were shown to be applicable to cultivar identification of non-PCNA accessions. We directly sequenced ast-linked fragments from 48 accessions with one-size peak of ast-linked fragment and found two distinctive groups of fragments based on single nucleotide polymorphisms. This result suggests that a bottleneck event occurred during ast allele development. We conclude that our fragment size profile can be used to accelerate PCNA breeding that uses non-PCNA parents and to study ast allele accumulation in persimmon.