Quantitative genotyping of CPCNA locus advances genetic strategies in PCNA persimmon breeding

Abstract

Persimmon (Diospyros kaki Thunb., 2n = 6x = 90) is a major fruit crop in eastern Asia, known for its unique in accumulating large amounts of proanthocyanidins (PAs) in the fruit flesh, resulting in strong astringency. The Pollination constant and non-astringent (PCNA) mutation, characterized by reduced PAs, allows for consumption before softening, akin to an apple. Two distinct types of PCNA persimmons, Japanese PCNA (J-PCNA) and Chinese PCNA (C-PCNA), exhibit different genetic regulatory mechanisms. The J-PCNA type is conferred by homozygous recessive alleles (ast), while C-PCNA possesses at least one dominant allele (CPCNA) at the CPCNA locus. The probability of generating PCNA individuals depends on the presence of ast or CPCNA alleles in parental plants. Previous research has quantified dosages of AST alleles in major Japanese cultivars, the status of AST alleles, especially CPCNA alleles, in Chinese cultivars remains undisclosed. This lack of understanding creates a significant obstacle in PCNA persimmon breeding efforts, particularly for the C-PCNA type. In this study, we developed a method to quantify the CPCNA locus by detecting the allele dosage of RO2, a marker strongly associated with CPCNA alleles across cultivars, using quantitative real-time PCR (qPCR). Our findings reveal that most C-PCNA cultivars and androecious germplasm exhibiting the PCNA trait contain only one CPCNA allele (Bbbbbb). Furthermore, we observed that the average ast allele dosage in Chinese genotypes was lower than that in Japanese non-PCNA genotypes. Quantifying the CPCNA locus assists in selecting PCNA candidate and also cultivating parents with sufficient CPCNA alleles (more than quadruple dose), ensuring that the offspring crossed with non-PCNA types inherit the PCNA trait. This approach will undoubtedly accelerate the breeding of novel PCNA cultivars and could also prevent inbreeding depression in PCNA breeding practices when crosses between J-PCNA cultivars. Our method provides crucial insights for enhancing PCNA persimmon breeding programs and advancing the development of improved cultivars.