Genomic analysis of fruit size and shape traits in apple: unveiling candidate genes through GWAS analysis

Abstract

Genomic tools facilitate the efficient selection of improved genetic materials within a breeding program. Here, we focus on two apple fruit quality traits: shape and size. We utilized data from 11 fruit morphology parameters gathered across three years of harvest from 355 genotypes of the apple REFPOP collection, which serves as a representative sample of the genetic variability present in European-cultivated apples. The data were then employed for genome-wide association studies (GWAS) using the FarmCPU and the BLINK models. The analysis identified 59 SNPs associated with fruit size and shape traits (35 with FarmCPU and 45 with BLINK) responsible for 71 QTNs. These QTNs were distributed across all chromosomes except for chromosomes 10 and 15. Thirty-four QTNs, identified by 27 SNPs, were related for size traits, and 37 QTNs, identified by 26 SNPs, were related to shape attributes. The definition of the haploblocks containing the most relevant SNPs served to propose candidate genes, among them the genes of the ovate family protein MdOFP17 and MdOFP4 that were in a 9.7kb haploblock on Chromosome 11. RNA-seq data revealed low or null expression of these genes in the oblong cultivar "Skovfoged" and higher expression in the flat "Grand'mere." The Gene Ontology enrichment analysis support a role of OFPs and hormones in shape regulation. In conclusion, this comprehensive GWAS analysis of the apple REFPOP collection has revealed promising genetic markers and candidate genes associated with apple fruit shape and size attributes, providing valuable insights that could enhance the efficiency of future breeding programs.

Figure 1

Spearman correlation analysis of fruit size and shape traits across multiple years, as well as their mean values across all years. The traits evaluated area (A), width mid-height (WMH), maximum width (MW), maximum height (MH) fruit shape index external I (FSII), distal fruit blockiness (DFB), fruit shape triangle (FST), proximal angle macro (PAMa), distal angle macro (DAMa), ellipsoid (E), circular (C), rectangular (R), eccentricity (ECC), and fruit shape internal (FSIINT). See correlation coefficients in Supplementary Fig. S2.

Figure 2

Summary of GWAS results for the size and shape traits using different models. QTNs obtained with Blink (x) and FarmCPU (□) models in the datasets for 2019 (●), 2020 (●), and mean values (●) are represented. The X-axis corresponds to the chromosomes, while the Y-axis represents the traits.

Figure 3

Violin plots displaying the frequency distribution of size (A) and shape (B) phenotypic values across genotypes. Each violin plot corresponds to a specific SNP-QTN combination, illustrating the distribution of trait values for different genotypes. In each violin plot, the X-axis represents the genotypes, with the first allele (on the left) indicating the homozygous genotype for the reference allele in the GDDH13 whole genome v1.1, the middle representing the heterozygous genotype, and the right side representing the homozygous genotype for the alternative or minor allele.

Figure 4

Linkage disequilibrium along Chromosome 11. The top figure displays Chromosome 11 from GDDH13v1.1, with position of markers published in this study and symbols representing QTNs found in this study (circles) and published (kites). The colors of the circles correspond to the associated QTN, and each circle is labeled with a letter representing the respective haploblock. Haploblocks, defined using GDDH13v1.1 positions, are as follows: Haploblock A: 4.661.534 to 4.958.286 (199 markers), Haploblock B: 5.930.883 to 5.948.789 pb (24 markers), Haploblock C: 9.046.670 to 9.556.662 pb (63 markers), Haploblock D: 12.638.696 to 13.198.304 pb (434 markers), Haploblock E: 14.355.224 to 14.402.233 pb (30 markers), and Haploblock F: 37.648.782 to 38.174.120 bp (241 markers). The color gradient from white to red represents the level of linkage disequilibrium (D′). A D′ value <20 is considered weak linkage disequilibrium, and red color indicates a D′ value of 100, representing strong linkage disequilibrium.

Figure 5

Integrated analysis of GWAS results, haploblocks, genotype–phenotype frequency, gene annotation, and RNA-seq, for FSIINT, CAT-own and Circular traits with the mean across years data. (A) GWAS results: this panel showcases multiple Manhattan plots representing the GWAS results for the three traits: FSIINT, CAT-own and circular. The plots depict the significance of genetic markers on each chromosome, colored based on the trait and the two models used (Blink and FarmCPU). Density plots illustrate SNP distribution on each chromosome. (B) Haploblocks & Haplotypes: the linkage disequilibrium (D′) based on the GDDH13v1 genome is presented. Haploblocks are identified using the criteria from Gabriel et al. [56], with colors indicating the strength of D′ (white = weak, red = strong). The haplotypes of each block and their allelic frequency are shown below. (C) Frequency Genotype–Phenotype: Allele frequency for the three traits is displayed, along with the corresponding apple shape genotypes (“Grand’mere” = flat, “Kansas Queen” = round, “Skovfoged” = oblong). (D) Gene annotation: This section presents the candidate genes annotated within the haploblock, utilizing the annotations from the HFTH1 whole genome v1.0. (E) RNA-seq: TPM data at the 13 DAA fruit stage for three candidate genes (HF43535, HF43536, and HF42456) across the three genotypes.

Figure 6

PhenoGram of the molecular markers on the physical map according to the apple GDDH13 whole genome v1.1. Significant markers mapped for apple fruit measures, including markers published in QTLs/QTNs analysis and the QTNs found in this study. Symbols: circle corresponds to “in this study” and kite, “significative markers of QTLs/QTNs published.” Color blue for size traits and green for shape traits. See details in Supplementary Figure S6.