(110) Suppression Subtractive Hybridization (SSH) Analysis for Detecting Genes Involved in Condensed Tannin Accumulation in Persimmon Fruit

Abstract

Pollination-constant and non-astringent (PCNA)-type persimmon has probably originated from astringent (non-PCNA)-type as a mutant that terminates condensed tannin accumulation at an early stage of fruit development. This trait is confirmed to be recessive and is controlled by a single locus. Since PCNA-type fruit stops tannin accumulation at an early stage, comparison of the gene expressions between PCNA- and non-PCNA-type will reveal the genes conferring condensed tannin accumulation in persimmon fruit. We performed suppression subtractive hybridization (SSH) analysis for detecting differentially expressed genes in non-PCNA-type fruit using BC1 offspring from a cross between PCNA `Fuyu' and non-PCNA “275-13” (F1 progeny derived from non-PCNA `Aizumishirazu' × PCNA `Taishu'). Fruits from seven individuals of PCNA or non-PCNA offspring in BC1 were sampled at early two stages of fruit development and total RNA was extracted by hot borate method from each fruit of different stage. Then, RNA was pooled as PCNA or non-PCNA bulk at two stages and cDNA was synthesized from each bulk for SSH analysis. A total of 5000 clones expressed differentially in non-PCNA-type fruit were picked from SSH library of two stages and 198 positive clones confirmed by differential screening were sequenced. The homologous sequences for the genes involved in flavonoid biosynthesis (CHS, CHI, F3H, F3'5'H, DFR, UFGT, and ANS) were obtained from the clones. The genes that are not considered to be involved in flavonoid biosynthesis so far (SCPL and DHQ) were also detected with high frequencies. We will discuss the role of these genes for condensed tannin accumulation in persimmon fruit.