Candidate gene analysis for ASTRINGENCY controlling fruit astringency in Diospyros kaki based on mRNA- and small RNA-sequencing analyses

The astringent taste caused by proanthocyanidins (PAs), which still remains at fruit maturation or harvesting stage, is a unique characteristic of persimmon. Pollination-constant and non-astringent (PCNA) type cultivars stop accumulating PAs in the early stage of fruit development, allowing the fruit to be edible without artificial de-astringency treatment. The PCNA trait is conferred by a recessive allele at a single locus, ASTRINGENCY (AST). The AST-locating genomic region (AST region) has been delimited in the polyploid D. kaki genome by comparative mapping, but the AST gene has yet to be identified. Here, we characterized a genomic region of diploid D. oleifera that corresponds to the AST region in D. kaki. Firstly, we obtained mRNA-seq data from siblings from a cross between ‘Taiten’ (non-PCNA) and ‘Kanshu’ (PCNA) and their parents at two fruit developmental stages in 2015 and at five stages in 2016. The genetic analysis concluded that there is no nonsynonymous substitution in the genic regions in the AST region. Differential expression analysis between PCNA and non-PCNA detected two differentially expressed genes (FDR <0.05) in the region, whereas their expression patterns were not correlated with PA accumulation during fruit development. We also sequenced small RNA from the same population as the differential expression analysis. Small RNAs were annotated in the D. oleifera genome, and 6 small RNAs in the AST region with significantly different normalized counts (p<0.05) between PCNA and non-PCNA were identified. These small RNAs were found to target 5 genes, whereas there were no effects in their mRNA expression levels. In conclusion, this study identified none of the promising AST candidates with clear difference between the astringent and non-astringent alleles. More detail information of polyploid D. kaki genome is required to obtain comprehensive set of candidate genes.