关键词: 甜瓜, 幼果果皮颜色, 基因定位, GR, MELO3C003375（CmAPRR2）

Abstract:

【Objective】 The aim of this study was to explore the inheritance pattern of immature rind color of melon (Cucumis melo L.), to fine map target gene GR, and to deepen the understanding of rind color change during fruit growth, so as to provide a guidance for the improvement of melon color with molecular design breeding.【Method】 The dark-green rind line ‘MR-1’ (C. melo ssp. agrestis) and the light-green rind line ‘LGR’ (C. melo ssp. melo) were used as parents to construct the F₁ hybrid population, and the BC₁F₁ backcross population was constructed from crossing by F₁ and ‘LGR’ for genetic analysis of immature rind color. By selecting 20 dark-green and light-green plants each in BC₁F₁ population, the DNA was mixed, respectively, and the BSA-seq was operated for initial mapping. Based on the resequencing of two parents, the molecular markers with better specificity were developed in the initial region and recombinant individuals were identified and selected to verify and narrow the interval for fine mapping the GR gene. By sequencing of coding regions between two parents according to the gene annotation, the candidate gene and key variant site were determined. Moreover, the phenotype of immature and mature fruit in the BC₁F₁ backcross population was investigated and assessed by correlation analysis to explore the underlying mechanism of rind color transition in the fruit development.【Result】 According to the investigation, the phenotype of all F₁ individual plants exhibited dark-green rind color. In addition, the immature rind color of BC₁F₁ backcross population was found to be separated, and the ratio of the number of dark-green to light-green was approximately 1﹕1. As well as, the ratio of the number of dark-green to light-green in the F₂ population was 3﹕1. These ratios corresponded to Mendel’s law of inheritance, indicating that the immature rind color of melon was a quality trait, controlled by a single nuclear gene GR, and the dark-green was dominant to light-green. Through BSA-seq, the gene was initially mapped to a 1.8 Mb interval on the long arm of chromosome 4. With developed molecular markers, 24 recombinant individuals were selected in expanded mapping population. The gene was further narrowed down to a region between markers 4-102 and 4-81 with a physical distance of 17.7 kb by genotype and phenotype verification of offspring, where were four predicted genes with latest annotation. Sequencing analysis revealed that a gene MELO3C003375 encoding GLKs transcription factor CmAPRR2 had several variations in ‘MR-1’ and ‘LGR’. Among them, there were three synonymous, a missense mutation, and a nonsense mutation. The nonsense mutation (G to T) appeared in the 856th base of the coding region led to premature translation termination and most of the Myb-DNA binding domain to be lost in ‘LGR’. Thus, the MELO3C003375 (CmAPRR2) was speculated to be the GR gene and the nonsense mutation was the key variation that affected the immature rind color of melon. It was found that there was a significant correlation between the rind color of immature fruit and mature fruit by investigating the phenotype of fruits in the BC₁F₁ backcross population.【Conclusion】 Immature rind color (dark green/light green) of melon was a quality trait and controlled by a single nuclear gene GR. By mapping, MELO3C003375 (CmAPRR2) was presumed to be the candidate gene that affected immature rind color.

Key words: melon, immature rind color, gene mapping, GR, MELO3C003375 (CmAPRR2)