Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) is an important cucurbit crop grown worldwide.  Watermelon fruit quality, fertility, and seed-setting rate are closely related to male flower development.  In this study, the different developmental stages of flower buds of the watermelon cultivar ‘Xinteda Zhengkang 9’ were distinguished by cytological observation, and transcriptome sequencing analysis was performed subsequently.  Acetocarmine staining of anthers was performed and the longitudinal and transverse diameters of the unopened male flower buds were measured.  Cytological observations of anthers at different developmental stages showed that the anther grew from the tetrad to the mature stage, and the longitudinal and transverse diameters of the flower buds increased.  The length of the male flower buds also changed significantly during development.  Transcriptome sequencing analysis at four periods, the tetrad (A group), mononuclear (B group), dikaryophase (C group), and mature stages (D group).  A total of 16 288 differentially expressed genes (DEGs) were detected in the four stages, with the prolongation of developmental stages, the number of DEGs increased gradually in the comparison groups, there was 2 014, 3 259, 4 628, 1 490, 3 495 and 1 132 DEGs revealed in six comparison groups (A-vs.-B, A-vs.-C, A-vs-D, B-vs.-C, B-vs.-D, and C-vs.-D), respectively.  Gene Ontology (GO) and KEGG enrichment analysis showed that the DEGs were mainly enriched in cellular component and starch and sucrose metabolism, phenylpropanoid biosynthesis and pentose sugar, etc.  Finally, we completely screened 59 DEGs in the six comparison groups, interestingly, we found one pollen-specific protein (Cla001608) that was significantly down-regulated (the value of log₂Fold Change up to 17.32), which indicated that it may play an important role in the development of male flowers.  This work provides insight into the molecular basis of the developmental stages of male flowers in watermelon and may aid in dominant cross breeding.

In vitro gynogenesis is an important tool used in haploid or homozygous double-haploid plant breeding.  However, because of low repeatability, embryoid induction rate and quality, the molecular mechanisms remain poorly understood.  Heat shock treatment can promote the transformation of the gametophytic pathway into the sporophyte pathway, which induces the occurrence of haploid.  In this study, unfertilized ovaries were heat shocked for 0 h (A0) before flowering and for 0 h (A1), 4 h (A3), 8 h (A5), 12 h (A7), and 24 h (A8), respectively, at 37°C at the first day of the flowering stage.  The ovule enlargement rate was increased from 0% at 25°C to 96.8% at 37°C (24 h treatment).  Thus, we aimed to investigate the gene expression patterns in unfertilized ovules of watermelon after different periods of heat shock by using RNA-Seq technology.  The results showed that compared with A3, A5, A7, and A8, the biosynthesis of amino acid, glycine, serine and threonine metabolic pathways in A1 has changed significantly.  This indicated that heat shock treatment affected the synthesis and transformation of amino acids during ovule expansion.  The transcriptome data suggested gene expressions of ovule growth were significantly changed by heat-specific influences.  The results provide new information on the complex relationship between in vitro gynogenesis and temperature.  This provides a basis for further study of the mechanism of heat shock affecting the expansion of watermelon ovule.