Malate accumulation in the vacuole largely determines apple (Malus domestica) fruit acidity, and low fruit acidity is strongly associated with truncation of Ma1, an ortholog of ALUMINUM-ACTIVATED MALATE TRANSPORTER9 (ALMT9) in Arabidopsis (Arabidopsis thaliana). A mutation at base 1,455 in the open reading frame of Ma1 leads to a premature stop codon that truncates the protein by 84 amino acids at its C-terminal end. Here, we report that both the full-length protein, Ma1, and its naturally occurring truncated protein, ma1, localize to the tonoplast; when expressed in Xenopus laevis oocytes and Nicotiana benthamiana cells, Ma1 mediates a malate-dependent inward-rectifying current, whereas the ma1-mediated transmembrane current is much weaker, indicating that ma1 has significantly lower malate transport activity than Ma1. RNA interference suppression of Ma1 expression in ‘McIntosh’ apple leaves, ‘Empire’ apple fruit, and ‘Orin’ apple calli results in a significant decrease in malate level. Genotyping and phenotyping of 186 apple accessions from a diverse genetic background of 17 Malus species combined with the functional analyses described above indicate that Ma1 plays a key role in determining fruit acidity and that the truncation of Ma1 to ma1 is genetically responsible for low fruit acidity in apple. Furthermore, we identified a C-terminal domain conserved in all tonoplast-localized ALMTs essential for Ma1 function; protein truncations into this conserved domain significantly lower Ma1 transport activity. We conclude that the truncation of Ma1 to ma1 reduces its malate transport function by removing a conserved C-terminal domain, leading to low fruit acidity in apple.

Acidity is a major contributor to fruit quality. Several organic acids are present in apple fruit, but malic acid is predominant and determines fruit acidity. The trait is largely controlled by the Malic acid (Ma) locus, underpinning which Ma1 that putatively encodes a vacuolar aluminum-activated malate transporter1 (ALMT1)-like protein is a strong candidate gene. We hypothesize that fruit acidity is governed by a gene network in which Ma1 is key member. The goal of this study is to identify the gene network and the potential mechanisms through which the network operates. Guided by Ma1, we analyzed the transcriptomes of mature fruit of contrasting acidity from six apple accessions of genotype Ma_ (MaMa or Mama) and four of mama using RNA-seq and identified 1301 fruit acidity associated genes, among which 18 were most significant acidity genes (MSAGs). Network inferring using weighted gene co-expression network analysis (WGCNA) revealed five co-expression gene network modules of significant (P < 0.001) correlation with malate. Of these, the Ma1 containing module (Turquoise) of 336 genes showed the highest correlation (0.79). We also identified 12 intramodular hub genes from each of the five modules and 18 enriched gene ontology (GO) terms and MapMan sub-bines, including two GO terms (GO:0015979 and GO:0009765) and two MapMap sub-bins (1.3.4 and 1.1.1.1) related to photosynthesis in module Turquoise. Using Lemon-Tree algorithms, we identified 12 regulator genes of probabilistic scores 35.5–81.0, including MDP0000525602 (a LLR receptor kinase), MDP0000319170 (an IQD2-like CaM binding protein) and MDP0000190273 (an EIN3-like transcription factor) of greater interest for being one of the 18 MSAGs or one of the 12 intramodular hub genes in Turquoise, and/or a regulator to the cluster containing Ma1. The most relevant finding of this study is the identification of the MSAGs, intramodular hub genes, enriched photosynthesis related processes, and regulator genes in a WGCNA module Turquoise that not only encompasses Ma1 but also shows the highest modular correlation with acidity. Overall, this study provides important insight into the Ma1-mediated gene network controlling acidity in mature apple fruit of diverse genetic background.

To unlock the power of next generation sequencing-based bulked segregant analysis in allele discovery in out-crossing woody species, and to understand the genetic control of the weeping trait, an F₁ population from the cross ‘Cheal’s Weeping’ × ‘Evereste’ was used to create two genomic DNA pools ‘weeping’ (17 progeny) and ‘standard’ (16 progeny). Illumina pair-end (2 × 151 bp) sequencing of the pools to a 27.1× (weeping) and a 30.4× (standard) genome (742.3 Mb) coverage allowed detection of 84562 DNA variants specific to ‘weeping’, 92148 specific to ‘standard’, and 173169 common to both pools. A detailed analysis of the DNA variant genotypes in the pools predicted three informative segregation types of variants: (type I) in weeping pool-specific variants, and (type II) and <hk×hk> (type III) in variants common to both pools, where the first allele is assumed to be weeping linked and the allele shown in bold is a variant in relation to the reference genome. Conducting variant allele frequency and density-based mappings revealed four genomic regions with a significant association with weeping: a major locus, Weeping (W), on chromosome 13 and others on chromosomes 10 (W2), 16 (W3), and 5 (W4). The results from type I variants were noisier and less certain than those from type II and type III variants, demonstrating that although type I variants are often the first choice, type II and type III variants represent an important source of DNA variants that can be exploited for genetic mapping in out-crossing woody species. Confirmation of the mapping of W and W2, investigation into their genetic interactions, and identification of expressed genes in the W and W2 regions provided insight into the genetic control of weeping and its expressivity in Malus.