Hand pollination is a necessary assisting method for pitaya (Selenicereus spp.) production to achieve a high yield. With the cultivated area increasing at an exponential rate in recent years, a comprehensive study of the pollination process was conducted. We developed an ideal medium for pitaya pollen in vitro germination in this study, then tested the activity of pollen collected from or stored for various time periods. We discovered that those collected between 2 h before blooming and 6 h after blooming had the higher germination rates (27.2–65.1%), the highest activity was at 2 h after blooming, and that storing them at 4°C for 24 hours reduces their germination rate from 65.2 percent to 35.5 percent and their production to about 82%. As a result, pollinating plants with pollen that has been held for more than 24 hours is not recommended unless a breakthrough in pollen storage is achieved. We also discovered that stigma receptivity and pollen activity are synchronized, which together determines the rate of fruit setting and the size of the fruit. Pollination within 6 hours after flowering offers the optimum fruit setting percentage and size, while pollination at 6:00 pm, 2 hours before blooming, is also a good alternative; however, pollination at 6:00 am the next morning is expected to result in a 23 % drop in productivity. These findings will be beneficial for reproductive biology research, as well as laying the groundwork for hand pollination to boost pitaya output and breeding efficiency.

Self-incompatibility (SI) is a major issue in dragon fruit (Selenicereus spp.) breeding and production. Therefore, a better understanding of the dragon fruit SI mechanism is needed to improve breeding efficiency and ultimate production costs. To reveal the underlying mechanisms of SI in dragon fruit, plant anatomy, de novo RNA sequencing-based transcriptomic analysis, and multiple bioinformatic approaches were used to analyze gene expression in the pistils of the self-pollinated and cross-pollinated dragon fruit flowers at different intervals of time after pollination. Using fluorescence microscopy, we observed that the pollen of ‘Hongshuijing’, a self-incompatible dragon fruit variety (S. monacanthus), germinated on its own stigma. However, the pollen tube elongation has ceased at 1/2 of the style, confirming that dragon fruit experiences gametophyte self-incompatibility (GSI). We found that the pollen tube elongation in vitro was inhibited by self-style glycoproteins in the SI variety, indicating that glycoproteins were involved in SI. That is to say the female S factor should be homologous of S-RNase or PrsS (P. rhoeas stigma S factor), both of which are glycoproteins and are the female S factors of the two known GSI mechanism respectively. Bioinformatics analyses indicated that among the 43,954 assembled unigenes from pistil, there were six S-RNase genes, while 158 F-box genes were identified from a pollen transcriptomic dataset. There were no P. rhoeas type S genes discovered. Thus, the identified S-RNase and F-box represent the candidate female and male S genes, respectively. Analysis of differentially expressed genes (DEGs) between the self and cross-pollinated pistils at different time intervals led to the identification of 6,353 genes. We then used a weighted gene co-expression network analysis (WGCNA) to find some non-S locus genes in SI responses in dragon fruit. Additionally, 13 transcription factors (TFs) (YABBY4, ANL2, ERF43, ARF2, BLH7, KNAT6, PIF3, two OBF1, two HY5 and two LHY/CCA) were identified to be involved in dragon fruit GSI. Thus, we uncovered candidate S and non-S genes and predicted more SI-related genes for a more detailed investigation of the molecular mechanism of dragon fruit SI. Our findings suggest that dragon fruit possesses a GSI system and involves some unique regulators. This study lays the groundwork for future research into SI mechanisms in dragon fruit and other plant species.

(1) Background: Wax gourd is an economically important vegetable crop in many tropical and sub-tropical countries in Asia. Fruit color is an important fruit quality trait, but the genetic, biochemical basis and regulatory network of fruit color variation in wax gourd are rarely studied. (2) Methods: In this study, two wax gourd inbred lines with different pericarp colors were used as materials to conduct joint metabolomic and transcriptomic analyses on mature fruit pericarp: B214 with yellow and B227 with dark green color. (3) Results: It was found that the chlorophyll content in the pericarp of B214 was significantly lower than that of B227, consistent with the down-regulation of several genes involved in the chlorophyll biosynthesis pathway, including hemA, hemB, hemC, hemF, chlH, chlI, chlM, POR, and CAO. The 229 metabolites showed differential accumulation levels between B214 and B227, and 4 anthocyanins, 5 flavanones, 25 flavones, 25 flavone C-glycosides, 12 flavonols, and 3 isoflavones were identified. In particular, cyanidin 3-O-glucoside, an anthocyanin contributing to the coloration of dark color, showed higher accumulation in B227 than in B214, probably due to the higher expression of genes of F3′H and glucosyl transferases (GTs) in B227. Transcription factors such as MYBs and bHLHs showed differential expressions between the two lines including bHLH14, a homolog of Arabidopsis AtbHLH14 that had significantly higher expression in B227 than in B214. bHLH14 was located in a region where the pericarp color (pc) locus was mapped, suggesting it may be a candidate gene for the pc locus. (4) Conclusions: This work supports the association of chlorophyll and flavonoid synthesis in wax gourd fruit color variation and also provides a good foundation for understanding the regulatory network for wax gourd coloration.

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