Genome-wide identification and expression analysis of the CmDMPgene family in melon (Cucumis melo L.)

【Objective】The application of haploid induction represents an effective approach for shortening breeding cycles in crop improvement programs. In several economically important cereals including maize and rice, particular genes belonging to the DMP (Domain of Unknown Function 679 Membrane Protein) family have been established as critical genetic components for generating haploid inducer lines. Despite its agricultural significance, melon (Cucumis melo L.) lacks a comprehensive investigation into its DMP gene family. To address this knowledge gap, the present study aims to perform a genome-wide identification of DMP homologs in the melon genome. Following their discovery, we will use an integrated approach of bioinformatics and molecular biology to characterize the fundamental protein properties, phylogenetic linkages with orthologs from related species, and tissue-specific transcrip-tion profiles of these genes. This multifaceted analysis seeks to elucidate the putative biological roles of the melon DMP family and establish a substantive basis for future functional studies.【Methods】The thick-skinned melon inbred line K7-2, characterized by robust growth and stable phenotypic traits, was employed as the experimental plant material. Comprehensive identification of CmDMP family members was performed through a genome-wide screening against the Melon (DHL92) v3.6.1 reference genome. Subsequently, the ExPASy online platform was employed to determine key physicochemical parameters of the encoded proteins, including amino acid count, molecular mass, theoretical isoelectric point, instability coefficient, hydrophobicity profile, and aliphatic index for each member; WoLFPSORT was employed for subcellular localization prediction; TBtools was used to generate gene chromosomal distribution maps; MEGA software was applied to construct a phylogenetic tree for evolutionary relationship analysis; MEME and InterPro databases were utilized to identify conserved motifs and domains, respectively; for the identification and categorization of cis- regulatory elements, the 2000- base pair region directly preceding the start codon was analyzed using the PlantCARE database. To clarify the gene expression patterns, root, stem, leaf, female flower, male flower tissues and pollen were collected separately. Total RNA was isolated with a commercial extraction kit and converted to complementary DNA (cDNA) following quality assessment. Gene-specific primers were constructed according to the coding sequences, and quantitative real-time PCR (RT-qPCR) amplification was carried out using the melon Actin gene for normalization. Each assay included three independent biological replicates, with four technical replicates per reaction. Relative expression levels were determined by the 2−ΔΔCt method. Statistical analysis of inter-group differences was performed using one-way ANOVA, supplemented by Tukey's test for multiple comparisons.【Results】Eight DMP family members were identified from the melon genome and named CmDMP1 to CmDMP8 according to their positions on the chromosomes. The proteins encoded by these genes exhibited diversity in basic physicochemical properties: the amino acid length ranged from 69 to 222, the molecular weight ranged from 7.85-24.54 kDa, and the theoretical isoelectric point distributed from 5.38 to 10.86. Stability analysis showed that except for CmDMP6, the instability indices of the other members were all below 40, indicating that they are stable proteins. Most proteins showed hydrophobic properties, while only CmDMP2 had a negative average hydrophilicity index, showing hydrophilic properties. All proteins had relatively similar aliphatic indices. Prediction of subcellular localization indicated that seven family members were likely to reside predominantly in the plasma membrane, and 1 member existed in both the cytoplasm and nucleus. Phylogenetic analysis clustered 28 DMP family members from melon, Arabidopsis thaliana, watermelon and cucumber into 4 subfamilies, with DMP family members distributed in each subfamily, indicating that DMP family members retained good cross- species conservation during evolution. Further conserved motif analysis revealed that except for CmDMP2, the other members contained multiple core motifs, and the composition and arrangement of motifs within the same subfamily were highly conserved. All members contained the DUF679 domain, but CmDMP2 only contained a superfamily domain, which was different from other members. Gene structure analysis indicated that there were 1-3 exons among the members. Analysis of promoter cis-acting elements found that the promoter regions of all genes contained light-responsive elements. Meanwhile, a large number of elements related to hormone responses (especially methyl jasmonate and abscisic acid) and abiotic stress responses (especially anaerobic induction) were detected. RT- qPCR results identified two genes (CmDMP1 and CmDMP7) highly expressed in melon pollen, which might play important roles in the development of melon haploid inducer lines; CmDMP4 was specifically expressed in roots; the expression levels of CmDMP5 and CmDMP6 in female flowers were significantly higher than those in other tissues; CmDMP2, CmDMP3, CmDMP4 and CmDMP6 showed almost no expression in pollen.【Conclusion】Eight DMP family genes were identified in melon in this study. These genes showed significant diversity in structure and physicochemical properties, retained cross-species conservation during evolution, and could respond to multiple hormone and abiotic stress signals. The results of tissue-specific expression indicated that CmDMP1 and CmDMP7 might be key genes for developing melon haploid inducer lines, while CmDMP4, CmDMP5 and CmDMP6 might regulate the growth and development of roots and female flowers, respectively. Subsequent studies can focus on the functional verification of CmDMP1 and CmDMP7 to explore their molecular pathways regulating melon haploid formation, providing theoretical support for understanding the mechanisms of melon reproductive development and haploid induction.