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.

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.

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.

Genetic Mapping and QTL Analysis of Stigma Color in Melon (Cucumis melo L.).

Melon is an important Cucurbitaceae crop. Field observations had shown that the green stigmas of melon are more attractive to pollinators than yellow stigmas. In this study, F2 and F2:3 populations obtained by crossing MR-1 (green stigma) and M4-7 (yellow stigma) were used for genetic analysis and mapping. A genetic map of 1,802.49 cm was constructed with 116 cleaved amplified polymorphism sequence (CAPS) markers. Two stable quantitative trait loci (QTLs) linked to the trait of stigma color were identified on chromosomes 2 (SC2.1) and 8 (SC8.1), respectively. An expanded F2 population was used to narrow down the confidence regions of SC2.1 and SC8.1. As a result, SC2.1 was further mapped to a 3.6 cm region between CAPS markers S2M3 and S2B1-3, explaining 9.40% phenotypic variation. SC8.1 was mapped to a 3.7-cm region between CAPS markers S8E7 and S8H-1, explaining 25.92% phenotypic variation. This study broadens our understanding of the mechanisms of stigma color regulation and will be of benefit to the breeding of melon.

Fine Mapping of Lycopene Content and Flesh Color Related Gene and Development of Molecular Marker-Assisted Selection for Flesh Color in Watermelon (Citrullus lanatus).

Lycopene content and flesh color are important traits determined by a network of carotenoid metabolic pathways in watermelon. Based on our previous study of genetic inheritance and initial mapping using F2 populations of LSW-177 (red flesh) × cream of Saskatchewan (pale yellow flesh), red flesh color was controlled by one recessive gene regulating red and pale yellow pigmentation, and a candidate region related to lycopene content was detected spanning a 392,077-bp region on chromosome 4. To obtain a more precise result for further study, three genetic populations and a natural panel of 81 watermelon accessions with different flesh colors were used in this research. Herein, we narrowed the preliminary mapping region to 41,233 bp with the linkage map generated from F2 populations of LSW-177 (red flesh) × cream of Saskatchewan (pale yellow flesh) with 1,202 individuals. Two candidate genes, Cla005011 and Cla005012, were found in the fine mapping region; therein Cla005011 was a key locus annotated as a lycopene ß-cyclase gene. Phylogenetic tree analysis showed that Cla005011 was the closest relative gene in gourd. LSW-177 × PI 186490 (white flesh) and another BC1 population derived from garden female (red flesh) × PI 186490 were generated to verify the accuracy of the red flesh candidate gene region. By analyzing the expression levels of candidate genes in different developmental stages of different color watermelon varieties, Cla005011 for the expression differences was not the main reason for the flesh color variation between COS and LSW-177. This indicated that the LCYB gene might regulate fruit color changes at the protein level. A new marker-assisted selection system to identify red and yellow flesh colors in watermelon was developed with flesh color-specific CAPS markers and tested in 81 watermelon accessions.