Molecular Markers and Marker-Assisted Selection Provide Genetic Insights for Identifying Key Quantitative Trait Locus for Watermelon Rind Thickness.

Rind thickness (RT) is an important agronomic trait in watermelon [Citrullus lanatus (Thunb.) Mansf.] and affects watermelon storability. However, genetic studies on this trait, as well as gene regulation studies, are scarce and of limited production significance. We constructed a temporary F2 generation using the highly differentiated thick-rind watermelon 'XiaoXiGua-4' and the thin-rind watermelon 'DuanMan' as parents and localized the Cla97C02G044120 gene, which controls the thickness of watermelon rind, to the intervals of chromosome 2, CL2-32303995 and CL2-32316840, through 2 years of genetic analysis. No exonic mutations were found in this gene, but two promoter mutations resulted in changes in the promoter progenitor. Fluorescence quantitative PCR analysis revealed highly significant differences in expression at 1 d and 28 d, and the expression was significantly lower in thick-skinned watermelon varieties. Marker-assisted selection (MAS) for this trait was performed using the Caps marker CL2-32303995 and the InDel marker CL2-32316840, which not only verified the stability of the localization interval but also distinguished thick rind from thin rind. These results can be used for germplasm resource screening and have strong breeding significance.

Clpf encodes pentatricopeptide repeat protein (PPR5) and regulates pink flesh color in watermelon (Citrullus lanatus L.).

KEY MESSAGE: The gene controlling pink flesh in watermelon was finely mapped to a 55.26-kb region on chromosome 6. The prime candidate gene, Cla97C06G122120 (ClPPR5), was identified through forward genetics. Carotenoids offer numerous health benefits; while, they cannot be synthesized by the human body. Watermelon stands out as one of the richest sources of carotenoids. In this study, genetic generations derived from parental lines W15-059 (red flesh) and JQ13-3 (pink flesh) revealed the presence of the recessive gene Clpf responsible for the pink flesh (pf) trait in watermelon. Comparative analysis of pigment components and microstructure indicated that the disparity in flesh color between the parental lines primarily stemmed from variations in lycopene content, as well as differences in chromoplast number and size. Subsequent bulk segregant analysis (BSA-seq) and genetic mapping successfully narrowed down the Clpf locus to a 55.26-kb region on chromosome 6, harboring two candidate genes. Through sequence comparison and gene expression analysis, Cla97C06G122120 (annotated as a pentatricopeptide repeat, PPR) was predicted as the prime candidate gene related to pink flesh trait. To further investigate the role of the PPR gene, its homologous gene in tomato was silenced using a virus-induced system. The resulting silenced fruit lines displayed diminished carotenoid accumulation compared with the wild-type, indicating the potential regulatory function of the PPR gene in pigment accumulation. This study significantly contributes to our understanding of the forward genetics underlying watermelon flesh traits, particularly in relation to carotenoid accumulation. The findings lay essential groundwork for elucidating mechanisms governing pigment synthesis and deposition in watermelon flesh, thereby providing valuable insights for future breeding strategies aimed at enhancing fruit quality and nutritional value.

Identification of Chromosomal Regions and Candidate Genes for Round leaf Locus in Cucumis melo L.

Leaf morphology plays a crucial role in plant classification and provides a significant model for studying plant diversity while directly impacting photosynthetic efficiency. In the case of melons, leaf shape not only influences production and classification but also represents a key genetic trait that requires further exploration. In this study, we utilized forward genetics to pinpoint a recessive locus, dubbed Cmrl (Round leaf), which is responsible for regulating melon leaf shape. Through bulked segregant analysis sequencing and extensive evaluation of a two-year F2 population, we successfully mapped the Cmrl locus to a 537.07 kb region on chromosome 8 of the melon genome. Subsequent genetic fine-mapping efforts, leveraging a larger F2 population encompassing 1322 plants and incorporating F2:3 phenotypic data, further refined the locus to an 80.27 kb interval housing five candidate genes. Promoter analysis and coding sequence cloning confirmed that one of these candidates, MELO3C019152.2 (Cmppr encoding a pentatricopeptide repeat-containing family protein, Cmppr), stands out as a strong candidate gene for the Cmrl locus. Notably, comparisons of Cmrl expressions across various stages of leaf development and different leaf regions suggest a pivotal role of Cmrl in the morphogenesis of melon leaves.

Deciphering the Genomic Characterization of the GGP Gene Family and Expression Verification of CmGGP1 Modulating Ascorbic Acid Biosynthesis in Melon Plants.

Ascorbic acid (AsA), also known as vitamin C, is a well-known antioxidant found in living entities that plays an essential role in growth and development, as well as in defensive mechanisms. GDP-L-galactose phosphorylase (GGP) is a candidate gene regulating AsA biosynthesis at the translational and transcriptional levels in plants. In the current study, we conducted genome-wide bioinformatic analysis and pinpointed a single AsA synthesis rate-limiting enzyme gene in melon (CmGGP1). The protein prediction analysis depicted that the CmGGP1 protein does not have a signaling peptide or transmembrane structure and mainly functions in the chloroplast or nucleus. The constructed phylogenetic tree analysis in multispecies showed that the CmGGP1 protein has a highly conserved motif in cucurbit crops. The structural variation analysis of the CmGGP1 gene in different domesticated melon germplasms showed a single non-synonymous type-base mutation and indicated that this gene was selected by domestication during evolution. Wild-type (WT) and landrace (LDR) germplasms of melon depicted close relationships to each other, and improved-type (IMP) varieties showed modern domestication selection. The endogenous quantification of AsA content in both the young and old leaves of nine melon varieties exhibited the major differentiations for AsA synthesis and metabolism. The real-time quantitative polymerase chain reaction (qRT-PCR) analysis of gene co-expression showed that AsA biosynthesis in leaves was greater than AsA metabolic consumption, and four putative interactive genes (MELO3C025552.2, MELO3C007440.2, MELO3C023324.2, and MELO3C018576.2) associated with the CmGGP1 gene were revealed. Meanwhile, the CmGGP1 gene expression pattern was noticed to be up-regulated to varying degrees in different acclimated melons. We believe that the obtained results would provide useful insights for an in-depth genetic understanding of the AsA biosynthesis mechanism, aimed at the development of improving crop plants for melon.

Molecular Mapping of Putative Genomic Regions Controlling Fruit and Seed Morphology of Watermelon.

The genetic regulatory basis of qualitative and quantitative phenotypes of watermelon is being investigated in different types of molecular and genetic breeding studies around the world. In this study, biparental F2 mapping populations were developed over two experimental years, and the collected datasets of fruit and seed traits exhibited highly significant correlations. Whole-genome resequencing of comparative parental lines was performed and detected single nucleotide polymorphism (SNP) loci were converted into cleaved amplified polymorphic sequence (CAPS) markers. The screened polymorphic markers were genotyped in segregating populations and two genetic linkage maps were constructed, which covered a total of 2834.28 and 2721.45 centimorgan (cM) genetic lengths, respectively. A total of 22 quantitative trait loci (QTLs) for seven phenotypic traits were mapped; among them, five stable and major-effect QTLs (PC-8-1, SL-9-1, SWi-9-1, SSi-9-1, and SW-6-1) and four minor-effect QTLs (PC-2-1 and PC-2-2; PT-2-1 and PT-2-2; SL-6-1 and SSi-6-2; and SWi-6-1 and SWi-6-2) were observed with 3.77-38.98% PVE. The adjacent QTL markers showed a good fit marker-trait association, and a significant allele-specific contribution was also noticed for genetic inheritance of traits. Further, a total of four candidate genes (Cla97C09G179150, Cla97C09G179350, Cla97C09G180040, and Cla97C09G180100) were spotted in the stable colocalized QTLs of seed size linked traits (SL-9-1 and SWi-9-1) that showed non-synonymous type mutations. The gene expression trends indicated that the seed morphology had been formed in the early developmental stage and showed the genetic regulation of seed shape formation. Hence, we think that our identified QTLs and genes would provide powerful genetic insights for marker-assisted breeding aimed at improving the quality traits of watermelon.

Clorf Encodes Carotenoid Isomerase and Regulates Orange Flesh Color in Watermelon (Citrullus lanatus L.).

Flesh color is a significant characteristic of watermelon. Although various flesh-color genes have been identified, the inheritance and molecular basis of the orange flesh trait remain relatively unexplored. In the present study, the genetic analysis of six generations derived from W1-1 (red flesh) and W1-61 (orange flesh) revealed that the orange flesh color trait was regulated by a single recessive gene, Clorf (orange flesh). Bulk segregant analysis (BSA) locked the range to ∼4.66 Mb, and initial mapping situated the Clorf locus within a 688.35-kb region of watermelon chromosome 10. Another 1,026 F2 plants narrowed the Clorf locus to a 304.62-kb region containing 32 candidate genes. Subsequently, genome sequence variations in this 304.62-kb region were extracted for in silico BSA strategy among 11 resequenced lines (one orange flesh and ten nonorange flesh) and finally narrowed the Clorf locus into an 82.51-kb region containing nine candidate genes. Sequence variation analysis of coding regions and gene expression levels supports Cla97C10G200950 as the most possible candidate for Clorf, which encodes carotenoid isomerase (Crtiso). This study provides a genetic resource for investigating the orange flesh color of watermelon, with Clorf malfunction resulting in low lycopene accumulation and, thus, orange flesh.

Comparative chloroplast genome analysis of seven extant Citrullus species insight into genetic variation, phylogenetic relationships, and selective pressure.

Citrullus ecirrhosus, Citrullus rehmii, and Citrullus naudinianus are three important related wild species of watermelon in the genus Citrullus, and their morphological differences are clear, however, their chloroplast genome differences remain unknown. This study is the first to assemble, analyze, and publish the complete chloroplast genomes of C. ecirrhosus, C. rehmii, and C. naudinianus. A comparative analysis was then conducted among the complete chloroplast genomes of seven extant Citrullus species, and the results demonstrated that the average genome sizes of Citrullus is 157,005 bp, a total of 130-133 annotated genes were identified, including 8 rRNA, 37 tRNA and 85-88 protein-encoding genes. Their gene content, order, and genome structure were similar. However, noncoding regions were more divergent than coding regions, and rps16-trnQ was a hypervariable fragment. Thirty-four polymorphic SSRs, 1,271 SNPs and 234 INDELs were identified. Phylogenetic trees revealed a clear phylogenetic relationship of Citrullus species, and the developed molecular markers (SNPs and rps16-trnQ) could be used for taxonomy in Citrullus. Three genes (atpB, clpP1, and rpoC2) were identified to undergo selection and would promote the environmental adaptation of Citrullus.

Integrated analysis of biparental and natural populations reveals CRIB domain-containing protein underlying seed coat crack trait in watermelon.

KEY MESSAGE: The scc locus of the watermelon seed coat crack trait was fine mapped on chromosome 3. Cla97C03G056110 (annotated as CRIB domain-containing protein) was regarded as the most likely candidate gene Seed coat crack (scc) is a special characteristic of watermelon compared with other cucurbit crops. However, information regarding the genetic basis of this trait is limited. We conducted a genetic analysis of six generations derived from PI 192938 (scc) and Cream of Saskatchewan (COS) (non-scc) parental lines and found that the scc trait was regulated by a single recessive gene through two years. Bulk segregant analysis sequencing (BSA-seq) and initial mapping placed the scc locus into an 808.8 kb region on chromosome 3. Evaluation of another 1152 F2 plants narrowed the scc locus to a 277.11 kb region containing 37 candidate genes. Due to the lack of molecular markers in the fine-mapping interval, we extracted the genome sequence variations in this 277.11 kb region with in silico BSA among seventeen re-sequenced lines (6 scc and 11 non-scc) and finally delimited the scc locus to an 8.34 kb region with only one candidate gene Cla97C03G056110 (CRIB domain-containing protein). Three single nucleotide polymorphism loci in the promoter region of Cla97C03G056110 altered cis-acting elements that were highly correlated with the nature watermelon panel. The expression of Cla97C03G056110 in seed coat tissue was higher in non-scc than in scc lines and was specifically expressed in seed coat compared with fruit flesh.

Whole-transcriptome analyses identify key differentially expressed mRNAs, lncRNAs, and miRNAs associated with male sterility in watermelon.

Male sterility is a valuable trait for watermelon breeding, as watermelon hybrids exhibit obvious heterosis. However, the underlying regulatory mechanism is still largely unknown, especially regarding the related non-coding genes. In the present study, approximately 1035 differentially expressed genes (DEGs), as well as 80 DE-lncRNAs and 10 DE-miRNAs, were identified, with the overwhelming majority down-regulated in male-sterile floral buds. Enrichment analyses revealed that the general phenylpropanoid pathway as well as its related metabolisms was predicted to be altered in a mutant compared to its fertile progenitor. Meanwhile, the conserved genetic pathway DYT1-TDF1-AMS-MS188-MS1, as well as the causal gene ClAMT1 for the male-sterile mutant Se18, was substantially disrupted during male reproductive development. In addition, some targets of the key regulators AMS and MS188 in tapetum development were also down-regulated at a transcriptional level, such as ABCG26 (Cla004479), ACOS5 (Cla022956), CYP703A2 (Cla021151), PKSA (Cla021099), and TKPR1 (Cla002563). Considering lncRNAs may act as functional endogenous target mimics of miRNAs, competitive endogenous RNA networks were subsequently constructed, with the most complex one containing three DE-miRNAs, two DE-lncRNAs, and 21 DEGs. Collectively, these findings not only contribute to a better understanding of genetic regulatory networks underlying male sterility in watermelon, but also provide valuable candidates for future research.

Identification of chromosome region and candidate genes for canary-yellow flesh (Cyf) locus in watermelon (Citrullus lanatus).

Genetic control of fruit flesh color in watermelon is complex, and significant knowledge gaps still exist. In the present study, we investigated the genetic basis of canary-yellow flesh color in watermelon inbred line PI 635597 using a segregating population derived from a cross between PI 635597 and another inbred line, Cream of Saskatchewan (pale yellow flesh color). We showed that a single dominant gene controls the canary-yellow flesh color for the Cyf (canary-yellow flesh) trait. Bulk segregant analysis (BSA) and fine genetic mapping narrowed down the Cyf locus to a 79.62-kb region on chromosome 6, which harbors 10 predicted genes. Sequence variation analysis in the promoter and coding regions and gene expression analysis in both parental lines and selected watermelon accessions with diverse fruit flesh colors support Cla97C06G122050 (unknown protein) and Cla97C06G122120 (pentatricopeptide repeat) as predicted candidate genes for the Cyf locus. Marker-assisted selection and sequence alignment showed that the Cyf locus could differentiate canary-yellow flesh and pale-yellow flesh. Our results indicate that the Cyf locus might be responsible for canary-yellow flesh color and carotenoid accumulation levels.

A recessive gene Cmpmr2F confers powdery mildew resistance in melon (Cucumis melo L.).

KEY MESSAGE: Identified a recessive gene (Cmpmr2F) associated with resistance to infection by the powdery mildew causing agent Podosphaera xanthii race 2F. Powdery mildew (PM) is one of the most destructive fungal diseases of melon, which significantly reduces the crop yield and quality. Multiple studies are being performed for in-depth genetic understandings of PM-susceptibility or -resistance mechanisms in melon plants, but the holistic knowledge of the precise genetic basis of PM-resistance is unexplored. In this study, we characterized the recessive gene "Cmpmr2F" and found its association with resistance against the PM causative agent "Podosphaera xanthii race 2F." Fine genetic mapping revealed the major-effect region of a 26.25-kb interval on chromosome 12, which harbored the Cmpmr2F gene corresponding to the MELO3C002403, encoding allantoate amidohydrolase. The functional gene annotation, expression pattern, and sequence alignment analyses were carried out using two contrast parent lines of melon "X055" PM-susceptible and "PI 124112" PM-resistant. Further, gene silencing of Cmpmr2F using virus-induced gene silencing (VIGS) significantly increased PM-resistance in the susceptible plant. In contrast to the previously reported studies, we identified that Cmpmr2F-silenced plants showed no impairment in growth due to less apparent negative effects in silenced melon plants. So, it is believed that the Cmpmr2F gene has great potential for further breeding studies to increase the P. xanthii race 2F resistance in melon. In short, our study provides new genetic resources and a solid foundation for further functional analysis of PM-resistance genes in melon, as well as powerful molecular markers for marker-assisted breeding aimed at developing new melon varieties resistant to PM infection.

The HD-ZIP Gene Family in Watermelon: Genome-Wide Identification and Expression Analysis under Abiotic Stresses.

Homeodomain-leucine zipper (HD-ZIP) transcription factors are one of the plant-specific gene families involved in plant growth and response to adverse environmental conditions. However, little information is available on the HD-ZIP gene family in watermelon. In this study, forty ClHDZs were systemically identified in the watermelon genome, which were subsequently divided into four distinctive subfamilies (I-IV) based on the phylogenetic topology. HD-ZIP members in the same subfamily generally shared similar gene structures and conserved motifs. Syntenic analyses revealed that segmental duplications mainly contributed to the expansion of the watermelon HD-ZIP family, especially in subfamilies I and IV. HD-ZIP III was considered the most conserved subfamily during the evolutionary history. Moreover, expression profiling together with stress-related cis-elements in the promoter region unfolded the divergent transcriptional accumulation patterns under abiotic stresses. The majority (13/23) of ClHDZs in subfamilies I and II were downregulated under the drought condition, e.g., ClHDZ4, ClHDZ13, ClHDZ18, ClHDZ19, ClHDZ20, and ClHDZ35. On the contrary, most HD-ZIP genes were induced by cold and salt stimuli with few exceptions, such as ClHDZ3 and ClHDZ23 under cold stress and ClHDZ14 and ClHDZ15 under the salt condition. Notably, the gene ClHDZ14 was predominantly downregulated by three stresses whereas ClHDZ1 was upregulated, suggesting their possible core roles in response to these abiotic stimuli. Collectively, our findings provide promising candidates for the further genetic improvement of abiotic stress tolerance in watermelon.

Novel Bisexual Flower Control Gene Regulates Sex Differentiation in Melon (Cucumis melo L.).

The sex-control system involves several mechanisms in melon. The present study identified a novel bisexual flower control gene from the hermaphroditic melon germplasm, different from the previously recognized one. Genetic analysis showed that a single recessive gene in the newly identified locus b controlled the bisexual flower phenotype in melons. We generated 1431 F2 segregating individuals for genetic mapping of locus b, which was delimited to a 47.94 kb region. Six candidate genes were identified in the delimited interval, and candidate No. 4 encoding melon CPR5 protein was selected as the suitable one for locus b and was denoted CmCPR5. CPR5 reportedly interacted with ethylene receptor ETR1 to regulate ethylene signal transduction. Moreover, the ethephon assays showed that the parental lines (unisexual line and bisexual line) had contrasting expression patterns of CmCPR5. The BiFC and LCI assays also confirmed that CmCPR5 interacted with CmETR1 in 0426 but not in Y101. However, crossover tests showed that CmETR1 functioned normally in both parental lines, suggesting CPR5 malfunction in Y101. This study proposed a corollary mechanism of bisexual flower regulation during stamen primordium development in which the inhibition of stamen primordia development was prevented by the malfunctioning CmCPR5, resulting in bisexual flowers.

H2O2 and Ca2+ Signaling Crosstalk Counteracts ABA to Induce Seed Germination.

Seed germination is a critical stage and the first step in the plant's life cycle. H2O2 and Ca2+ act as important signal molecules in regulating plant growth and development and in providing defense against numerous stresses; however, their crosstalk in modulating seed germination remains largely unaddressed. In the current study, we report that H2O2 and Ca2+ counteracted abscisic acid (ABA) to induce seed germination in melon and Arabidopsis by modulating ABA and gibberellic acid (GA3) balance. H2O2 treatment induced a Ca2+ influx in melon seeds accompanied by the upregulation of cyclic nucleotide-gated ion channel(CNGC) 20, which encodes a plasma membrane Ca2+-permeable channel. However, the inhibition of cytoplasmic free Ca2+ elevation in the melon seeds and Arabidopsis mutant atcngc20 compromised H2O2-induced germination under ABA stress. CaCl2 induced H2O2 accumulation accompanied by the upregulation of respiratory burst oxidase homologue(RBOH) D and RBOHF in melon seeds with ABA pretreatment. However, inhibition of H2O2 accumulation in the melon seeds and Arabidopsis mutant atrbohd and atrbohf abolished CaCl2-induced germination under ABA stress. The current study reveals a novel mechanism in which H2O2 and Ca2+ signaling crosstalk offsets ABA to induce seed germination. H2O2 induces Ca2+ influx, which in turn increases H2O2 accumulation, thus forming a reciprocal positive-regulatory loop to maintain a balance between ABA and GA3 and promote seed germination under ABA stress.

Transcriptomic Analysis of Fusarium oxysporum Stress-Induced Pathosystem and Screening of Fom-2 Interaction Factors in Contrasted Melon Plants.

Fusarium wilt is one of the most destructive and less controllable diseases in melon, which is usually caused by fusarium oxysporum. In this study, transcriptome sequencing and Yeast Two-Hybrid (Y2H) methods were used for quantification of differentially expressed genes (DEGs) involved in fusarium oxysporum (f. sp. melonis race 1) stress-induced mechanisms in contrasted melon varieties (M4-45 "susceptible" and MR-1 "resistant"). The interaction factors of Fom-2 resistance genes were also explored in response to the plant-pathogen infection mechanism. Transcriptomic analysis exhibited total 1,904 new genes; however, candidate DEGs analysis revealed a total of 144 specific genes (50 upregulated and 94 downregulated) for M4-45 variety and 104 specific genes (71 upregulated and 33 downregulated) for MR-1 variety, respectively. The analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway depicted some candidate DEGs, including Phenylalanine metabolism, phenylpropane biosynthesis, plants-pathogen interaction, and signal transduction of plant hormones, which were mainly involved in disease resistance metabolic pathways. The weighted gene co-expression network analysis (WGCNA) analysis revealed a strong correlation module and exhibited the disease resistance-related genes encoding course proteins, transcription factors, protein kinase, benzene propane biosynthesis path, plants-pathogen interaction pathway, and glutathione S-transferase. Meanwhile, the resistance-related specific genes expression was relatively abundant in MR-1 compared to the M4-45, and cell wall-associated receptor kinases (MELO3C008452 and MELO3C008453), heat shock protein (Cucumis_melo_newGene_172), defensin-like protein (Cucumis_melo_newGene_5490), and disease resistance response protein (MELO3C016325), activator response protein (MELO3C021623), leucine-rich repeat receptor protein kinase (MELO3C024412), lactyl glutathione ligase (Cucumis_melo_newGene_36), and unknown protein (MELO3C007588) were persisted by exhibiting the upregulated expressions. At the transcription level, the interaction factors between the candidate genes in response to the fusarium oxysporum induced stress, and Y2H screening signified the main contribution of MYB transcription factors (MELO3C009678 and MELO3C014597), BZIP (MELO3C011839 and MELO3C019349), unknown proteins, and key enzymes in the ubiquitination process (4XM334FK014). The candidate genes were further verified in exogenously treated melon plants with f. oxysporum (Fom-2, Race 1), Abscisic acid (ABA), Methyl Jasmonite (MeJA), and Salicylic acid (SA), using the fluorescence quantitative polymerase chain reaction (qRT-PCR) analysis. The overall expression results indicated that the SA signal pathway is involved in effective regulation of the Fom-2 gene activity.

Identification and Characterization Roles of Phytoene Synthase (PSY) Genes in Watermelon Development.

Phytoene synthase (PSY) plays an essential role in carotenoid biosynthesis. In this study, three ClPSY genes were identified through the watermelon genome, and their full-length cDNA sequences were cloned. The deduced proteins of the three ClPSY genes were ranged from 355 to 421 amino acid residues. Phylogenetic analysis suggested that the ClPSYs are highly conserved with bottle gourd compared to other cucurbit crops PSY proteins. Variation in ClPSY1 expression in watermelon with different flesh colors was observed; ClPSY1 was most highly expressed in fruit flesh and associated with the flesh color formation. ClPSY1 expression was much lower in the white-fleshed variety than the colored fruits. Gene expression analysis of ClPSY genes in root, stem, leaf, flower, ovary and flesh of watermelon plants showed that the levels of ClPSY2 transcripts found in leaves was higher than other tissues; ClPSY3 was dominantly expressed in roots. Functional complementation assays of the three ClPSY genes suggested that all of them could encode functional enzymes to synthesize the phytoene from Geranylgeranyl Pyrophosphate (GGPP). Some of the homologous genes clustered together in the phylogenetic tree and located in the synteny chromosome region seemed to have similar expression profiles among different cucurbit crops. The findings provide a foundation for watermelon flesh color breeding with regard to carotenoid synthesis and also provide an insight for the further research of watermelon flesh color formation.

Comparative Transcriptome Analysis Identified Key Pathways and Genes Regulating Differentiated Stigma Color in Melon (Cucumis melo L.).

Stigma color is an important morphological trait in many flowering plants. Visual observations in different field experiments have shown that a green stigma in melons is more attractive to natural pollinators than a yellow one. In the current study, we evaluated the characterization of two contrasted melon lines (MR-1 with a green stigma and M4-7 with a yellow stigma). Endogenous quantification showed that the chlorophyll and carotenoid content in the MR-1 stigmas was higher compared to the M4-7 stigmas. The primary differences in the chloroplast ultrastructure at different developmental stages depicted that the stigmas of both melon lines were mainly enriched with granum, plastoglobulus, and starch grains. Further, comparative transcriptomic analysis was performed to identify the candidate pathways and genes regulating melon stigma color during key developmental stages (S1-S3). The obtained results indicated similar biological processes involved in the three stages, but major differences were observed in light reactions and chloroplast pathways. The weighted gene co-expression network analysis (WGCNA) of differentially expressed genes (DEGs) uncovered a "black" network module (655 out of 5302 genes), mainly corresponding to light reactions, light harvesting, the chlorophyll metabolic process, and the chlorophyll biosynthetic process, and exhibited a significant contribution to stigma color. Overall, the expression of five key genes of the chlorophyll synthesis pathway-CAO (MELO03C010624), CHLH (MELO03C007233), CRD (MELO03C026802), HEMA (MELO03C011113), POR (MELO03C016714)-were checked at different stages of stigma development in both melon lines using quantitative real time polymerase chain reaction (qRT-PCR). The results exhibited that the expression of these genes gradually increased during the stigma development of the MR-1 line but decreased in the M4-7 line at S2. In addition, the expression trends in different stages were the same as RNA-seq, indicating data accuracy. To sum up, our research reveals an in-depth molecular mechanism of stigma coloration and suggests that chlorophyll and related biological activity play an important role in differentiating melon stigma color.