A cryptic variation in a member of the Ovate Family Proteins is underlying the melon fruit shape QTL fsqs8.1

Abstract

The gene underlying the melon fruit shape QTL fsqs8.1 is a member of the Ovate Family Proteins. Variation in fruit morphology is caused by changes in gene expression likely due to a cryptic structural variation in this locus. Melon cultivars have a wide range of fruit morphologies. Quantitative trait loci (QTL) have been identified underlying such diversity. This research focuses on the fruit shape QTL fsqs8.1, previously detected in a cross between the accession PI 124112 (CALC, producing elongated fruit) and the cultivar 'Piel de Sapo' (PS, producing oval fruit). The CALC fsqs8.1 allele induced round fruit shape, being responsible for the transgressive segregation for this trait observed in that population. In fact, the introgression line CALC8-1, carrying the fsqs8.1 locus from CALC into the PS genetic background, produced perfect round fruit. Following a map-based cloning approach, we found that the gene underlying fsqs8.1 is a member of the Ovate Family Proteins (OFP), CmOFP13, likely a homologue of AtOFP1 and SlOFP20 from Arabidopsis thaliana and tomato, respectively. The induction of the round shape was due to the higher expression of the CALC allele at the early ovary development stage. The fsqs8.1 locus showed an important structural variation, being CmOFP13 surrounded by two deletions in the CALC genome. The deletions are present at very low frequency in melon germplasm. Deletions and single nucleotide polymorphisms in the fsqs8.1 locus could not be not associated with variation in fruit shape among different melon accessions, what indicates that other genetic factors should be involved to induce the CALC fsqs8.1 allele effects. Therefore, fsqs8.1 is an example of a cryptic variation that alters gene expression, likely due to structural variation, resulting in phenotypic changes in melon fruit morphology.

Fig. 1

High-resolution mapping of fsqs8.1. A schematic representation of the reference genome V4.0 (Castanera et al. 2020) based on the double haploid line DHL92 is depicted in the upper part of the figure, indicating the position of markers, annotated genes and the repetitive sequence rich region. The length of the genes and the direction of the transcription are indicated with arrows. The deduced genome of CALC8-1 based on re-sequencing data is depicted in the lower part. Blue dashed lines indicates the large deletions observed in CALC8-1. The position of fsqs8.1 based on the fine mapping experiments is indicated below the CALC8-1 genome. The candidate MELO3C025206 is highlighted in green. On the right, typical fruits of PI 124,112 (CALC, donor parent), PS (recipient parent) and CALC8-1 (introgression line carrying the fsqs8.1 locus from CALC in PS background) are also shown) (color figure online)

Fig. 2

Developmental analysis and q-PCR of SC8-3, PS and CALC8-1 ovaries. a Ovaries from the three genotypes at anthesis. b Ovary length, width (both in µm) and shape index for the three genotypes through ovary development stages: E1 (carpel primordial initiated), E2 (primordial stigmas appear), E3 (stigma and style defined and anthers with pollen) and E4 (ovule primordial initiated). Asterisks (*) indicates means of CALC8-1 or SC8-3 ovaries statistically significant different (p < 0.05) from PS means c Accumulation of MELOC025206 transcripts estimated by qRT-PCR between genotypes and developmental stages. Different letters indicate statistically significant differences among genotypes within developmental stages (p < 0.05). d Paraffin sections of representative ovaries from the three genotypes at E4

Fig. 3

Phylogeny of Arabidopsis thaliana, tomato and melon ovate family protein genes based on Poisson distances (Zuckerkandl and Pauling 1965). Bootstrap values were based on 2000 replicates. Evolutionary analyses were conducted in MEGA X (Kumar et al. 2018). In order to simplify the figure, the nodes that do not include informative members for the current report are collapsed. The full phylogenetic tree is depicted in Fig. S2

Fig. 4

Haplotypes found for CmOFP13 among 1175 re-sequenced melon accessions (Zhao et al. 2019). a SNPs defining the four haplotypes and their position on the melon genome v4.0 (Castanera et al. 2020). CALC8-1 shares the same haplotype as PI 124,112 (c), while PS shows the B haplotype. b Distribution of the four haplotypes through accessions belonging to different world regions

Fig. 5

Alteration of organ shape due to overexpression of CmOFP13 in Arabidopsis thaliana. a Images of A. thaliana wild-type plants (Col0) and transgenic plants expressing CmOFP13 from CALC8-1 showing a mild (L1 and L8) or strong (L6 and L11) phenotype at the 9-day-old seedling stage, adult rosette, flowers, pistils, and mature siliques. Scale bars are depicted in each stage. b Comparison between ovary length, width and the length/width ratio between Col-0 and L6 transgenic line. (*) Indicates statistically significant differences (p < 0.001)

Fig. 6

Interactions of fsqs8.1 in different genetic situations. a Comparison of CALC8-1, SC8-3 and CALC8-1-x-SC8-3 hybrid fruit shape indexes. The blue bar indicates the expected mid-parent mean, and the asterisk the significance of the contrast (p = 0.0002). b Interaction between a and fsqs8.1 in an F₂ population from the cross between CALC8-1 and ‘Mucha Nesvi’ (MN, a monoecious traditional cultivar). The means for the four homozygous allele combinations for the two loci (CALC/MN) for fsqs8.1, ANDRO/MONO for andromonoecious/monoecious (a), are shown on the left and the significance of the gene effects and interactions according two-way ANOVA on the right. c The fruit shape index of fruits from hybrids between accessions belonging to different melon horticultural groups with PS and CALC8-1 is depicted on the left. On the right, the significance of the effects (GB, genetic background, fsqs8.1 PS or CALC8-1 allele at the locus, and the two years) and their interactions from the three-way ANOVA, are shown