A novel gene, screened by cDNA-AFLP approach, contributes to lowering the acidity of fruit in apple

cDNA-AFLP, coupled with bulked segregant analysis (BSA), was used to screen genes expressed differently between low- and high-acid apple fruits from hybrids of ‘Toko’ × ‘Fuji’ (Malus × domestica Borkh.). Sixty-four combinations of AFLP primers produced 2240 fragments, of which only one showed different expression between low- and high-acid fruits. The specific fragment was cloned and sequenced, and the complete cDNA was achieved by 3′ and 5′ rapid amplification of cDNA ends (RACE). The screened gene, designated as Mal-DDNA (GenBank accession no. DQ417661), showed no significant homology to clones in GenBank. The relatedness between fruit acidity and the transcription level of Mal-DDNA was identified by RT-PCR analysis on 30 hybrids. RT-PCR analysis indicated that Mal-DDNA transcripted in low-acid fruits at both early and ripe stages whereas in high- and mid-acid fruits, it did not transcript at the early stage. RNA gel-blot hybridization indicated that Mal-DDNA transcripted only in fruits and had clear difference between low- and high/mid-acid fruits. There was a good indication that Mal-DDNA existed as one copy in apple genome by Southern blot. Possible regulation of Mal-DDNA in apple fruit acidity is also discussed in the paper.