Simple sequence repeats (SSRs) are widely used in mapping constructions and comparative and genetic diversity analyses. Here, 103,056 SSR loci were found in Cucurbita species by in silico PCR. In general, the frequency of these SSRs decreased with the increase in the motif length, and di-nucleotide motifs were the most common type. For the same repeat types, the SSR frequency decreased sharply with the increase in the repeat number. The majority of the SSR loci were suitable for marker development (84.75% in Cucurbita moschata, 94.53% in Cucurbita maxima, and 95.09% in Cucurbita pepo). Using these markers, the cross-species transferable SSR markers between C. pepo and other Cucurbitaceae species were developed, and the complicated mosaic relationships among them were analyzed. Especially, the main syntenic relationships between C. pepo and C. moschata or C. maxima indicated that the chromosomes in the Cucurbita genomes were highly conserved during evolution. Furthermore, 66 core SSR markers were selected to measure the genetic diversity in 61 C. pepo germplasms, and they were divided into two groups by structure and unweighted pair group method with arithmetic analysis. These results will promote the utilization of SSRs in basic and applied research of Cucurbita species.

Melon,Cucumis meloL., is an important horticultural crop with abundant morphological variability, but the genetic diversity and relationships within wild and cultivated melons remain unclear to date. In this study, thick-skinned (TC) (cultivated subspeciesmelo), thin-skinned (TN) (cultivated subspeciesagrestis), and wild accessions were analyzed for genetic diversity and relationships using 36 microsatellite markers. A total of 314 alleles were detected with a mean allelic number of 8.72 and polymorphism information content of 0.67. Cluster analysis of the accessions resulted in four distinct clusters (I, II, III, and IV) broadly matching with the TC, TN, and wild groups. Cluster I contained only two Indian wild accessions. Cluster II was consisted of 49 South Asian accessions, 34 wild accessions, and 15 TN accessions. Cluster III was a typical TC group including 51 multiorigin TC accessions and one wild accession. The remaining 88 accessions, including 75 TN accessions, 6 wild accessions, and 7 TC accessions, formed the cluster IV, and all the TN and wild accessions in this cluster were from China. These findings were also confirmed by Principal component analysis and STRUCTURE analysis. The South Asian subspeciesagrestisaccessions, wild and cultivated, had close genetic relationships with a distinctive genetic background. Chinese wild melons showed closeness to cultivated subspeciesagrestislandraces and could be a return from the indigenous cultivated melons. The AMOVA and pairwise F statistics (FST) presented genetic differentiation among the three groups, with the strongest differentiation (FST= 0.380) between TC and TN melons. These results offer overall information on genetic diversity and affiliations within a variety of melon germplasms and favor efficient organization and utilization of these resources for the current breeding purpose.

Leaf is a vital organ of plants that plays an essential role in photosynthesis and respiration. As an important agronomic trait in leaf development, leaf shape is classified into lobed, entire (no-lobed), and serrated in most crops. In this study, two-lobed leaf watermelon inbred lines WT2 and WCZ, and a no-lobed leaf watermelon inbred line WT20 were used to create two F₂ populations. Segregation analysis suggested that lobed leaves were dominant over the no-lobed leaves, and it was controlled by a signal gene. A locus on watermelon chromosome 4 controlling watermelon lobed/no-lobed leaves was identified through BSA-seq strategy combined with linkage analysis. The candidate gene was fine-mapped to a 61.5 kb region between 21,224,481 and 21,285,957 bp on watermelon chromosome 4 using two F₂ populations. Four functional genes were annotated in the candidate region, while sequences blast showed that there was a single-base deletion (A/-) only in the exon of Cla018360, which resulted in premature termination of translation in the no-lobed leaf lines. Function prediction showed that Cla018360 encodes an HD-Zip protein that has been reported to regulate the development of leaf shape. The single-base deletion also occurred in the HD-Zip domain. We inferred that the Cla018360 gene is the candidate gene for regulating the development of lobed/no-lobed leaves in watermelon. Gene expression analysis showed that Cla018360 was highly expressed in young leaves. Phylogenetic analysis showed that Cla018360 had a close genetic relationship with AtHB51, which had been reported to regulate the formation of leaf shape in Arabidopsis. Furthermore, transcriptome analysis showed that a total of 333 differentially expressed genes were identified between WT2 and WT20, of which 115 and 218 genes were upregulated and downregulated in no-lobed leaved watermelon WT20. This study not only provides a good entry point for studying leaf development but also provides foundational insights into breeding for special plant architecture in watermelon.

Rind pattern is one of the most important appearance qualities of watermelon, and the mining of different genes controlling rind pattern can enrich the variety of consumer choices. In this study, a unique intermittent rind stripe was identified in the inbred watermelon line WT20. The WT20 was crossed with a green stripe inbred line, WCZ, to construct F₂ and BC₁ segregating populations and to analyze the genetic characterization of watermelon stripe. Genetic analysis showed that the intermittent stripe was a qualitative trait and controlled by a single dominant gene, ClIS. Fine mapping based on linkage analysis showed that the ClIS gene was located on the 160 Kb regions between 25.92 Mb and 26.08 Mb on watermelon chromosome 6. Furthermore, another inbred watermelon line with intermittent stripe, FG, was re-sequenced and aligned on the region of 160 Kb. Interestingly, only two SNP variants (T/C, A/T) were present in both WT20 and FG inbred lines at the same time. The two SNPs are located in 25,961,768 bp (T/C) and 25,961,773 bp (A/T) of watermelon chromosome 6, which is located in the promoter region of Cla019202. We speculate that Cla019202 is the candidate gene of ClIS which controls the intermittent stripe in watermelon. In a previous study, the candidate gene ClGS was proved to control dark green stripe in watermelon. According to the verification of the two genes ClIS and ClGS in 75 watermelon germplasm resources, we further speculate that the ClGS gene may regulate the color of watermelon stripe, while the ClIS gene regulates the continuity of watermelon stripe. The study provides a good entry point for studying the formation of watermelon rind patterns, as well as providing foundation insights into the breeding of special appearance quality in watermelon.

The early flowering and less lateral branches in watermelon hold significant agricultural value. The synergistic effects of these traits provide an ideal template for watermelon plant architecture improvement. However, the molecular regulatory networks underlying the development of lateral organs (including branches and flowers) in watermelon remain unclear. In this study, we found ClTFL1 knockout lines significantly promote flowering time and inhibit lateral branching and tendril formation, while also leading to a mild apical flower phenotype. These findings indicate that the function of ClTFL1 in watermelon is more extensive than that of its homologous genes in Arabidopsis, rice, and tomato. Through yeast two-hybrid screening, we identified the interacting proteins of ClTFL1, including members of the 14-3-3 family ClGRF8, ClGRF9, and ClGRF12. Bimolecular fluorescence complementation (BiFC) assays further demonstrated ClTFL1 could directly interact with the ClGRF8, ClGRF9, and ClGRF12 protein. The knockout of ClGRF8 and ClGRF12 leads to reduced lateral branches and early flowering. These phenotypes are highly consistent with those of ClTFL1 knockout mutants. Our findings demonstrate the important role of the ClTFL1-ClGRFs module in regulating lateral organ development and flowering time in watermelon, offering important targets for watermelon plant architectural modification and molecular breeding.