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Summary: The firmness of fleshy fruit crops has a significant effect on their quality, consumer preference, shelf life and transportability. In a combined quantitative trait locus and genome‐wide association studies study of apple fruit texture, we identified a mutation (C‐G) in the ethylene response factor‐associated amphiphilic repression (EAR) motif in the coding region of the apple ETHYLENE RESPONSE FACTOR4 (ERF4) gene. Chromatin immunoprecipitation sequencing showed that ERF4 binds to the promoter of ERF3, which is involved in regulation of ethylene biosynthesis. The EAR mutation in ERF4 results in reduced repression of ERF3 expression, which is turn promotes ethylene production and loss of fruit firmness. ERF4 acts as a transcriptional repressor whose activity is modulated by a TOPLESS co‐repressor 4 (TPL4)‐binding EAR repression motif. Biolayer interferometry analysis showed that the mutation in the EAR motif causes a reduction in the interaction with TPL4. Suppression of ERF4 or TPL4 promoted fruit ripening and ethylene production. Taken together, our results provide insights into how ERF4 allelic variation underlies an important fruit quality trait. Significance Statement: Fruit softening is a hallmark of ripening in apple fruit, but the degree of softening varies among cultivars, with corresponding variation in their quality and consumer preference. Much attention has been paid to the molecular characterization of fruit softening; however, the underlying genetic mechanisms are still not well understood. Here, we show that a mutation in the seqeucene encoding the EAR motif of ERF4 results in a decrease in its repression activity, thereby promoting fruit softening and ripening. [ABSTRACT FROM AUTHOR]

Acidity levels greatly affect the taste and flavor of fruit, and consequently its market value. In mature apple fruit, malic acid is the predominant organic acid. Several studies have confirmed that the major quantitative trait locus Ma largely controls the variation of fruit acidity levels. The Ma locus has recently been defined in a region of 150 kb that contains 44 predicted genes on chromosome 16 in the Golden Delicious genome. In this study, we identified two aluminum-activated malate transporter-like genes, designated Ma1 and Ma2, as strong candidates of Ma by narrowing down the Ma locus to 65-82 kb containing 12-19 predicted genes depending on the haplotypes. The Ma haplotypes were determined by sequencing two bacterial artificial chromosome clones from G.41 (an apple rootstock of genotype Mama) that cover the two distinct haplotypes at the Ma locus. Gene expression profiling in 18 apple germplasm accessions suggested that Ma1 is the major determinant at the Ma locus controlling fruit acidity as Ma1 is expressed at a much higher level than Ma2 and the Ma1 expression is significantly correlated with fruit titratable acidity ( R = 0.4543, P = 0.0021). In the coding sequences of low acidity alleles of Ma1 and Ma2, sequence variations at the amino acid level between Golden Delicious and G.41 were not detected. But the alleles for high acidity vary considerably between the two genotypes. The low acidity allele of Ma1, Ma1- 1455A, is mainly characterized by a mutation at base 1455 in the open reading frame. The mutation leads to a premature stop codon that truncates the carboxyl terminus of Ma1-1455A by 84 amino acids compared with Ma1-1455G. A survey of 29 apple germplasm accessions using marker CAPS that targets the SNP in Ma1 showed that the CAPS allele was associated completely with high pH and highly with low titratable acidity, suggesting that the natural mutation-led truncation is most likely responsible for the abolished function of Ma for low pH or high acidity in apple. [ABSTRACT FROM AUTHOR]

Tree architecture is an important, complex and dynamic trait affected by diverse genetic, ontogenetic and environmental factors. 'Wijcik McIntosh', a columnar (reduced branching) sport of 'McIntosh' and a valuable genetic resource, has been used intensively in apple-breeding programs for genetic improvement of tree architecture. The columnar growth habit is primarily controlled by the dominant allele of gene Co ( columnar) on linkage group-10. But the Co locus is not well mapped and the Co gene remains unknown. To precisely map the Co locus and to identify candidate genes of Co, a sequence-based approach using both peach and apple genomes was used to develop new markers linked more tightly to Co. Five new simple sequence repeats markers were developed (C1753-3520, C18470-25831, C6536-31519, C7223-38004 and C7629-22009). The first four markers were obtained from apple genomic sequences on chromosome-10, whereas the last (C7629-22009) was from an unanchored apple contig that contains an apple expressed sequence tag CV082943, which was identified through synteny analysis between the peach and apple genomes. Genetic mapping of these five markers in four F populations of 528 genotypes and 290 diverse columnar selections/cultivars (818 genotypes in total) delimited the Co locus in a genetic interval with 0.37 % recombination between markers C1753-3520 and C7629-22009. Marker C18470-25831 co-segregates with Co in the 818 genotypes studied. The Co region is estimated to be 193 kb and contains 26 predicted gene in the 'Golden Delicious' genome. Among the 26 genes, three are putative LATERAL ORGAN BOUNDARIES (LOB) DOMAIN (LBD) containing transcription factor genes known of essential roles in plant lateral organ development, and are therefore considered as strong candidates of Co, designated MdLBD1, MdLBD2, and MdLBD3. Although more comprehensive studies are required to confirm the function of MdLBD1- 3, the present work represents an important step forward to better understand the genetic and molecular control of tree architecture in apple. [ABSTRACT FROM AUTHOR]