Identification of candidate genes for malic acid content in tomato fruits and development of associated SNP markers

Malic acid is one of the major organic acids in tomato ( Solanum lycopersicum L.) fruit and plays a critical role in flavor and overall fruit quality. Identifying genes associated with malic acid accumulation and elucidating their genetic mechanisms can provide valuable insights for tomato quality improvement. In this study, a genome-wide association study (GWAS) was conducted to identify candidate genes and single nucleotide polymorphism (SNP) loci associated with malic acid content in tomato fruit. A natural population consisting of 200 advanced-generation tomato inbred lines was used as the experimental material, and malic acid content was measured at the red-ripening stage. High-quality SNPs obtained through DNA resequencing and variant detection were used for GWAS to identify loci associated with variation in fruit malic acid content. The GWAS identified 90 significant SNP loci, all located on chromosome 6. Linkage disequilibrium (LD) block analysis and candidate gene annotation revealed 17 genes within the associated regions. Based on Pfam and Swiss-Prot database annotation and literature review, five candidate genes, Solyc06g072910.2, Solyc06g072920.3, Solyc06g072950.3, Solyc06g072960.1, and Solyc06g073050.2, were identified as being associated with malic acid content. These genes encode members of the aluminum-activated malate transporter (ALMT) family, ABCB transporter family, and NAC domain-containing proteins. Nine SNP variants were detected within the five candidate genes. Primers were developed for six selected SNP loci, and genotyping was performed using the penta-primer amplification refractory mutation system (PARMS). Correlation analysis between SNP genotypes and malic acid phenotypes demonstrated a strong association, confirming that these loci are significantly related to malic acid content in tomato fruit. The molecular markers developed in this study provide valuable tools for marker-assisted selection, and the identified candidate genes establish a foundation for further investigation into malic acid biosynthesis and metabolic regulation in tomato.