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Acidity is a critical component determining apple fruit quality. Previous studies reported two major acidity quantitative trait loci (QTLs) on linkage groups (LGs) 16 ( Ma ) and 8 ( Ma3 ), respectively, and their homozygous genotypes  mama  and  ma3ma3  usually confer low titratable acidity (TA) (<3.0 mg ml −1 ) to apple fruit. However, apples of genotypes  Ma - ( MaMa  and  Mama ) or  Ma3 - ( Ma3Ma3  and  Ma3ma3 ) frequently show an acidity range spanning both regular (TA 3.0–10.0 mg ml −1 ) and high (TA > 10 mg ml −1 ) acidity levels. To date, the genetic control for high-acidity apples remains essentially unknown. In order to map QTLs associated with high acidity, two genomic DNA pools, one for high acidity and the other for regular acidity, were created in an interspecific F 1  population Royal Gala ( Malus domestica ) × PI 613988 ( M. sieversii ) of 191 fruit-bearing progenies. By Illumina paired-end sequencing of the high and regular acidity pools, 1,261,640 single-nucleotide variants (SNVs) commonly present in both pools were detected. Using allele frequency directional difference and density (AFDDD) mapping approach, one region on chromosome 4 and another on chromosome 6 were identified to be putatively associated with high acidity, and were named  Ma6  and  Ma4 , respectively. Trait association analysis of DNA markers independently developed from the  Ma6  and  Ma4  regions confirmed the mapping of  Ma6  and  Ma4 . In the background of  MaMa , 20.6% of acidity variation could be explained by  Ma6 , 28.5% by  Ma4,  and 50.7% by the combination of both. The effects of  Ma6  and  Ma4  in the background of  Mama  were also significant, but lower. These findings provide important genetic insight into high acidity in apple.

Identification of two QTLs associated with high fruit acidity in apple using pooled genome sequencing analysis