Photosynthesis serves as the primary source of nutrients synthesized in higher plants, and improving photosynthetic efficiency can significantly increase crop yield and fruit quality. Leaf color mutants represent ideal materials for studying chloroplast development and photosynthesis mechanisms and have been widely characterized in field crops. However, relevant research on watermelon leaf color mutants remains scarce. In this study, we isolated a yellow-green phenotype mutant, PKH352, from an EMS-mutagenized watermelon mutant library. The chlorophyll content and maximal photochemical efficiency in PKH352 were significantly decreased. Genetic analysis showed that the mutated trait was controlled by a single nuclear gene, which was named Clygp (Citrullus lanatus yellow-green plant). Through MutMap and linkage analysis in an F₂ population of 440 plants, we identified a single nucleotide polymorphism (SNP) mutation within ClG42_04g0106300, which encoded a signal recognition particle 54 kDa protein, as the causal variant for the yellow-green phenotype. Further validation using a CRISPR/Cas9-mediated system confirmed that knockout of ClG42_04g0106300 results in the yellow-green phenotype in watermelon. In addition, comparative transcriptomic analysis revealed that mutations in ClG42_04g0106300 greatly affected the expression of key genes associated with chloroplast development and photosynthesis, providing strong evidence that this gene plays a critical role in these biological pathways. Taken together, these findings provide insights into the molecular mechanisms underlying chloroplast development and photosynthetic efficiency, offering a theoretical basis for breeding watermelon varieties with high photosynthetic efficiency.

Genotyping by Target Sequencing (GBTS) technology, known for its flexibility, high efficiency, high throughput, and low cost, has been increasingly employed in molecular breeding. However, there is still limited study on the design and development of high-throughput genotyping tools in watermelon. In this study, we identified 112000 high quality SNPs by analyzing the resequencing data of 43 cultivated watermelon accessions. 11921 and 6094 SNPs were selected for developing two sets of watermelon liquid-phase chips with different marker densities, named Watermelon 10K and 5K, respectively. Furthermore, the SNPs and Indels of most mapped gene/QTLs for many agronomic important traits in watermelon were also integrated into the two chips for foreground selection. These chips have been tested using GBTS technology in various applications in watermelon. The genotyping of 76 accessions by Watermelon 5K liquid-phase chip showed an average detection rate of 99.28 % and 81.78 % for cultivated and wild watermelon accessions, respectively. This provided enough markers information for GWAS and two significant QTLs, ssc1.1 and ssc1.2, associated with soluble sugar content were detected. Furthermore, BSA-seq analysis for non-lobed leaf and dwarf traits were validated by liquid-phase chips, and the candidate region was consistent with our previous studies. Additionally, we precisely introduced the Cldw1 and Clbl genes into an elite inbred line WT2 using Watermelon 5K for assisted selection, resulting in the development of three new germplasm with good plant architecture. As a high-throughput genotyping liquid-phase SNP array, the Watermelon 10K and 5K chips will greatly facilitate functional studies and molecular breeding in watermelon.

The color and pattern of watermelon rind are crucial external traits that directly affect consumer preferences. Watermelons with stripes having a stronger color than the background rind are ideal for studying stripe patterns in plants, while there is still limited knowledge about the genetic mechanisms underlying stripe coloration due to the lack of germplasm resources. In this study, we focused on a watermelon germplasm with colorless stripes, and genetic analysis revealed that the trait is controlled by a single recessive gene. The gene Clsc (Citrullus lanatus stripe coloration), which is responsible for the colorless stripe, was localized into a 147.6 kb region on Chr9 by linkage analysis in a large F₂ mapping population. Further analysis revealed that the Cla97C09G175170 gene encodes the APRR2 transcription factor, plays a crucial role in determining the watermelon colorless stripe phenotype and was deduced to be related to chlorophyll synthesis and chloroplast development. Physiological experiments indicated that Cla97C09G175170 may significantly influence chloroplast development and chlorophyll synthesis in watermelon. The results of this study provide a better understanding of the molecular mechanism of stripe coloration in watermelon and can be useful in the development of marker-assisted selection (MAS) for new watermelon cultivars.

Pruning is time-consuming and laborious in watermelon cultivation, which can not meet the needs for simplified cultivation in the future. The development of branchless lines will provide important germplasms for breeding watermelon varieties and is an important method for genetic improvement. In this study, the watermelon accession, Wu Cha Zao (WCZ) is a branchless inbred line that carries the branchless gene Clbl, which was used as the donor parent to develop branchless near isogenic lines (NILs). To construct the NILs of Clbl, WCZ crossed with the normal branching watermelon inbred line WT20 which was used as the recurrent parent. The co-segregating markers dCAPS10 and Indel1 with Clbl were used for foreground selection, and a total of 108 SSR markers was selected with good polymorphism between two parental lines for background selection which had relatively uniform distribution across 11 chromosomes. Using these markers to select individuals from the BC₁F₁, BC₂F₁, and BC₂F₂ generations, three NILs with a proportion of recurrent parent genome (PRPG) > 99% were finally obtained. The lateral branch and plant height phenotypes did not significantly differ between the NILs and WCZ, indicating that the NILs of Clbl under the genetic background of WT20 has been successfully developed. These results provide ideal materials for further in-depth analysis of the genetic mechanisms of lateral branch development and ideal plant architecture breeding in watermelon.