Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been difficult and costly. The whole genome sequencing with next-generation sequencing (NGS) technologies provides large amounts of sequence data to develop numerous microsatellite markers at whole genome scale. SSR markers have great advantage in cross-species comparisons and allow investigation of karyotype and genome evolution through highly efficient computation approaches such as in silico PCR. Here we described genome wide development and characterization of SSR markers in the watermelon (Citrullus lanatus) genome, which were then use in comparative analysis with two other important crop species in the Cucurbitaceae family: cucumber (Cucumis sativus L.) and melon (Cucumis melo L.). We further applied these markers in evaluating the genetic diversity and population structure in watermelon germplasm collections. A total of 39,523 microsatellite loci were identified from the watermelon draft genome with an overall density of 111 SSRs/Mbp, and 32,869 SSR primers were designed with suitable flanking sequences. The dinucleotide SSRs were the most common type representing 34.09 % of the total SSR loci and the AT-rich motifs were the most abundant in all nucleotide repeat types. In silico PCR analysis identified 832 and 925 SSR markers with each having a single amplicon in the cucumber and melon draft genome, respectively. Comparative analysis with these cross-species SSR markers revealed complicated mosaic patterns of syntenic blocks among the genomes of three species. In addition, genetic diversity analysis of 134 watermelon accessions with 32 highly informative SSR loci placed these lines into two groups with all accessions of C.lanatus var. citorides and three accessions of C. colocynthis clustered in one group and all accessions of C. lanatus var. lanatus and the remaining accessions of C. colocynthis clustered in another group. Furthermore, structure analysis was consistent with the dendrogram indicating the 134 watermelon accessions were classified into two populations. The large number of genome wide SSR markers developed herein from the watermelon genome provides a valuable resource for genetic map construction, QTL exploration, map-based gene cloning and marker-assisted selection in watermelon which has a very narrow genetic base and extremely low polymorphism among cultivated lines. Furthermore, the cross-species transferable SSR markers identified herein should also have practical uses in many applications in species of Cucurbitaceae family whose whole genome sequences are not yet available.

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.

Plant trichome development is influenced by diverse developmental and environmental signals, but the molecular mechanisms involved are not well understood in most plant species. Fruit spines (trichomes) are an important trait in cucumber (Cucumis sativus L.), as they affect both fruit smoothness and commercial quality. Spine Base Size1 (CsSBS1) has been identified as essential for regulating fruit spine size in cucumber. Here, we discovered that CsSBS1 controls a season-dependent phenotype of spine base size in wild-type plants. Decreased light intensity led to reduced expression of CsSBS1 and smaller spine base size in wild-type plants, but not in the mutants with CsSBS1 deletion. Additionally, knockout of CsSBS1 resulted in smaller fruit spine base size and eliminated the light-induced expansion of spines. Overexpression of CsSBS1 increased spine base size and rescued the decrease in spine base size under low light conditions. Further analysis revealed that ELONGATED HYPOTCOTYL5(HY5), a major transcription factor involved in light signaling pathways, directly binds to the promoter of CsSBS1 and activates its expression. Knockout of CsHY5 led to smaller fruit spine base size and abolished the light-induced expansion of spines. Taken together, our study findings have clarified a CsHY5-CsSBS1 regulatory module that mediates light-regulated spine expansion in cucumber. This finding offers a strategy for cucumber breeders to develop fruit with stable appearance quality under changing light conditions.