Fruit characteristics of sweet watermelon are largely the result of human selection. Here we report an improved watermelon reference genome and whole-genome resequencing of 414 accessions representing all extant species in theCitrullusgenus. Population genomic analyses reveal the evolutionary history ofCitrullus, suggesting independent evolutions inCitrullus amarusand the lineage containingCitrullus lanatusandCitrullus mucosospermus. Our findings indicate that different loci affecting watermelon fruit size have been under selection during speciation, domestication and improvement. A non-bitter allele, arising in the progenitor of sweet watermelon, is largely fixed inC. lanatus. Selection for flesh sweetness started in the progenitor ofC. lanatusand continues through modern breeding on loci controlling raffinose catabolism and sugar transport. Fruit flesh coloration and sugar accumulation might have co-evolved through shared genetic components including a sugar transporter gene. This study provides valuable genomic resources and sheds light on watermelon speciation and breeding history.

How raffinose (Raf) family oligosaccharides, the major translocated sugars in the vascular bundle in cucurbits, are hydrolyzed and subsequently partitioned has not been fully elucidated. By performing reciprocal grafting of watermelon (Citrullus lanatus) fruits to branch stems, we observed that Raf was hydrolyzed in the fruit of cultivar watermelons but was backlogged in the fruit of wild ancestor species. Through a genome-wide association study, the alkaline alpha-galactosidase ClAGA2 was identified as the key factor controlling stachyose and Raf hydrolysis, and it was determined to be specifically expressed in the vascular bundle. Analysis of transgenic plants confirmed that ClAGA2 controls fruit Raf hydrolysis and reduces sugar content in fruits. Two single-nucleotide polymorphisms (SNPs) within the ClAGA2 promoter affect the recruitment of the transcription factor ClNF-YC2 (nuclear transcription factor Y subunit C) to regulate ClAGA2 expression. Moreover, this study demonstrates that C. lanatus Sugars Will Eventually Be Exported Transporter 3 (ClSWEET3) and Tonoplast Sugar Transporter (ClTST2) participate in plasma membrane sugar transport and sugar storage in fruit cell vacuoles, respectively. Knocking out ClAGA2, ClSWEET3, and ClTST2 affected fruit sugar accumulation. Genomic signatures indicate that the selection of ClAGA2, ClSWEET3, and ClTST2 for carbohydrate partitioning led to the derivation of modern sweet watermelon from non-sweet ancestors during domestication.

The gene ClLCYB contributes to the red flesh color of domesticated watermelon through decreased protein level to convert lycopene to β-carotene in the carotenog

Abscisic acid (ABA) is a critical regulator of seed development and germination. β-glucosidases (BGs) have been suggested to be contributors to increased ABA content because they catalyze the hydrolysis of ABA-glucose ester to release free ABA. However, whether BGs are involved in seed development is unclear. In this study, a candidate gene,ClBG1, in watermelon was selected for targeted mutagenesis via the CRISPR/Cas9 system. Seed size and weight were significantly reduced in theClbg1-mutant watermelon lines, which was mainly attributed to decreased cell number resulting from decreased ABA levels. A transcriptome analysis showed that the expression of 1015 and 1429 unique genes was changed 10 and 18 days after pollination (DAP), respectively. Cytoskeleton- and cell cycle-related genes were enriched in the differentially expressed genes of wild type andClbg1-mutant lines during seed development. Moreover, the expression of genes in the major signaling pathways of seed size control was also changed. In addition, seed germination was promoted in theClbg1-mutant lines due to decreased ABA content. These results indicate thatClBG1may be critical for watermelon seed size regulation and germination mainly through the modulation of ABA content and thereby the transcriptional regulation of cytoskeleton-, cell cycle- and signaling-related genes. Our results lay a foundation for dissecting the molecular mechanisms of controlling watermelon seed size, a key agricultural trait of significant economic importance.

Carotenoid pigments confer photoprotection and visual attraction and serve as precursors for many important signaling molecules. Herein, the orange-fruited phenotype of a tomato elite inbred line resulting from sharply reduced carotenoid levels and an increased β-carotene-to-lycopene ratio in fruit was shown to be controlled by a single recessive gene, oft3. BSA-Seq combined with fine mapping delimited the oft3 gene to a 71.23 kb interval on chromosome 4, including eight genes. Finally, the oft3 candidate gene SlIDI1, harboring a 116 bp deletion mutation, was identified by genome sequence analysis. Further functional complementation and CRISPR–Cas9 knockout experiments confirmed that SlIDI1 was the gene underlying the oft3 locus. qRT–PCR analysis revealed that the expression of SlIDI1 was highest in flowers and fruit and increased with fruit ripening or flower maturation. SlIDI1 simultaneously produced long and short transcripts by alternative transcription initiation and alternative splicing. Green fluorescent protein fusion expression revealed that the long isoform was mainly localized in plastids and that an N-terminal 59-amino acid extension sequence was responsible for plastid targeting. Short transcripts were identified in leaves and fruit by 5’ RACE and in fruit by 3’ RACE, which produced corresponding proteins lacking transit peptides and/or putative peroxisome targeting sequences, respectively. In SlIDI1 mutant fruit, SlBCH1 transcription involved in β-carotenoid catabolism was obviously suppressed, which may be responsible for the higher β-carotene-to-lycopene ratio and suggested potential feedback regulatory mechanisms involved in carotenoid pathway flux.