Map-based Cloning And Functional Analysis Of Fruit Skin Netting-Related Gene CmSN In Melon

Melon is a principal crop in the Cucurbitaceae family,extensively cultivated and of significant economic value.As a leading melon producer globally,China plays a pivotal role in the fruit and vegetable production sector.Notably,netted melons,with their distinct reticulated patterns on mature fruit,enhance aesthetic appeal and storage durability,thereby boosting their commercial value.The skin netting（SN）is a crucial visual quality trait of melons.While the physiological,cytological,and genetic aspects of its formation have been studied,the key genes regulating SN formation remain unidentified.Therefore,exploring the critical genes controlling melon SN,elucidating the molecular mechanisms underlying fruit netting characteristics,and identifying the fundamental causes of phenotypic differences between netted and smooth-skinned melons are of essential scientific and practical importance for improving melon’s visual quality.This study aims to isolate the key gene regulating melon netting（SN）using positional cloning techniques,and to explore its variation in natural populations along with its characteristics during domestication.Additionally,the research verifies the gene’s function through transient expression and heterologous transformation experiments,and employs protein interaction analysis to elucidate the molecular mechanisms involved in the regulation of melon SN formation.The main research findings are as follows:1.Using the smooth-skinned melon H906 and netted melon H581 as parental lines,we created an F₂/F_2:3segregating population and performed statistical analyses on the fruit netting phenotype under various environmental conditions.Genetic analysis revealed that melon fruit netting is controlled by a single dominant gene.Utilizing a high-density genetic map previously established by our research team,the melon SN gene was initially mapped to chromosome 2 with a genetic distance of 0.72 c M and a physical distance of approximately 351 kb.We finely mapped the CmSN gene to a 71 kb region,which contains five candidate genes.Further sequence alignment analysis showed that only the promoter and coding regions of MELO3C010288exhibited variations,and it shares the closest phylogenetic relationship with Cucurbitaceae Eam A family members,displaying specific expression in skin tissues.Therefore,MELO3C010288 is proposed to be the key candidate gene responsible for melon netting and is designated as CmSN.2.Using whole-genome re-sequencing data from 175 natural melon populations,GWAS was conducted on melons to pinpoint CmSN within the 16,300 kb to 16,500 kb region（approximately 200 kb）.The results showed that the overlapping segment between GWAS and linkage mapping,including the lead SNP(SNP^16,408,669)in the region with the strongest GWAS signal,matched the linkage mapping findings.Analysis with F_STand XP-CLR indicated that the CmSN locus is under strong domestication selection in the long-melon subspecies（ssp.melo）.In contrast,no selection signal was observed in the short-melon subspecies（ssp.agrestis）,suggesting CmSN may be a crucial region linked to domestication.Additionally,we converted the non-synonymous mutation SNP^16,408,669of the CmSN gene into a CAPS marker,which can differentiate between smooth-skinned and netted melons in natural populations.3.To elucidate the genetic function of CmSN,VIGS silencing and transient overexpression were performed on different melons.The results indicate that transient overexpression of CmSN can delay the formation of fruit netting.Upon VIGS injection into smooth-skinned melon fruits,the silenced plants exhibited raised areas and cell suberization on the fruit skin.Furthermore,CmSN was heterologously overexpressed in tomatoes（Ailsa Craig）,resulting in fruits with rough skin and significantly higher lignin content in their skin compared to control fruits.These findings indicate that the CmSN gene can influence the formation of melon netting.4.Using online software,an in-depth predictive analysis was conducted on the core promoter region,transcription factor binding sites,and regulatory elements of the CmSN gene.Dual-luciferase reporter assays were utilized to construct promoter report gene vectors with varying segment deletions,and experiments were performed to assess the impact of these deletions on promoter activity.This led to the identification of SNP^16410605as the core promoter region of the CmSN gene.5.Using methods such as bioinformatics and transcriptome sequencing,the transcription factors Cmb ZIP30 and Cm NAC53 were predicted to regulate the CmSN promoter.Results from yeast one-hybrid and LUC reporter assays showed that Cmb ZIP30 and Cm NAC53 can bind to the promoter of the CmSN and activate its transcription.Additionally,Cm NAC53 directly activates the transcription of Cmb ZIP30.The interaction between Cmb ZIP30 and Cm NAC53 at the protein level reduces the transcriptional activation of the CmSN.Transient transformation experiments in melon fruit further confirmed that Cm NAC53 can bind with Cmb ZIP30 to the CmSN promoter and activate its expression,thereby reducing netting formation.These findings elucidate the cooperative regulation of CmSN by Cm NAC53 and Cmb ZIP30 in the formation of melon netting.In conclusion,CmSN has been recognized as a crucial gene affecting the development of netting on melon fruits.Cm NAC53 can directly bind to the promoters of Cmb ZIP30 and CmSN to activate their expression,thus inhibiting netting on melon fruits.This research not only sheds new light on the molecular mechanisms behind melon netting formation but also offers innovative approaches and tools for enhancing melon quality and breeding advancements.