Identification and Characterization of Known and Novel MicroRNAs in Five Tissues of Wax Gourd (Benincasa hispida) Based on High-Throughput Sequencing

MicroRNAs (miRNAs) are endogenous single-stranded non-coding small RNAs of 20-24 nucleotides and play important roles in many plant biological and metabolic processes. Wax gourd is an important vegetable of Cucurbitacea family, with great economic and medicinal value. Although miRNAs have been extensively studied in model plant species, less is known in wax gourd (Benincasa hispida). In this study, in order to identify miRNAs in wax groud, five independent small RNA libraries were constructed using leaf, root, stem, flower, and fruit of B227. Based on high-throughput Illumina deep sequencing. In total, 422 known and 409 novel miRNAs were identified from five libraries. Comparative analysis revealed that many miRNAs were differentially expressed among different tissues, indicating tissue-specific expression of some miRNAs. qRT-PCR verified the reliability of small RNA sequencing results. Furthermore, miRNAs with similar expression patterns among five tissues were clustered into the same profile, among which many miRNAs were found with relatively high expression in the fruit of wax gourd. MiR164-x had the highest expression in fruit than in other tissues and many NAC transcription factors were predicted as its target genes. We propose that miR164 might regulate fruit development by forming miR164-NAC module in wax gourd. Taken together, this study provides the first global miRNAs profiling of wax gourd, and lays the foundation for understanding the regulatory roles of miRNAs in the growth and development processes of wax gourd.

Compatibility Evaluation and Anatomical Observation of Melon Grafted Onto Eight Cucurbitaceae Species

Melon (Cucumis melo) is one of the top 10 fruits in the world, and its production often suffers due to soil-borne diseases. Grafting is an effective way to solve this problem. However, graft incompatibility between scion and rootstock limits the application of melon grafting. In this study, the melon was grafted onto eight Cucurbitaceae species (cucumber, pumpkin, melon, luffa, wax gourd, bottle gourd, bitter gourd, and watermelon), and graft compatibility evaluation and anatomical observation were conducted. Taking melon homo-grafted plants as control, melon grafted onto cucumber and pumpkin rootstocks was compatible, while melon grafted onto luffa, wax gourd, bottle gourd, bitter gourd, and watermelon rootstocks was incompatible based on the scion dry weight on day 42 after grafting. Meanwhile, we found that starch–iodine staining of scion stem base is an index to predict graft compatibility earlier, on day 14 after grafting. Further, microsection observations showed that there was more cell proliferation at graft junction of melon hetero-grafted combinations; vascular reconnection occurred in all graft combinations. However, excess callose deposited at graft junction resulted in the blockage of photosynthate transport, thus, leading to starch accumulation in scion stem base, and finally graft incompatibility. In addition, undegraded necrotic layer fragments were observed at graft junctions of melon grafted onto incompatible bitter gourd and watermelon rootstocks. The above results provide clues for the selection and breeding of compatible Cucurbitaceae rootstocks of melon and demonstrate that starch accumulation in scion base and callose deposition at graft junction is associated with melon graft compatibility.

Identification and Application of BhAPRR2 Controlling Peel Colour in Wax Gourd (Benincasa hispida)

Peel color is an important factor affecting commodity quality in vegetables; however, the genes controlling this trait remain unclear in wax gourd. Here, we used two F2 genetic segregation populations to explore the inheritance patterns and to clone the genes associated with green and white skin in wax gourd. The F2 and BC1 trait segregation ratios were 3:1 and 1:1, respectively, and the trait was controlled by nuclear genes. Bulked segregant analysis of both F2 plants revealed peaks on Chr5 exceeding the confidence interval. Additionally, 6,244 F2 plants were used to compress the candidate interval into a region of 179 Kb; one candidate gene, Bch05G003950 (BhAPRR2), encoding two-component response regulator-like protein Arabidopsis pseudo-response regulator2 (APRR2), which is involved in the regulation of peel color, was present in this interval. Two bases (GA) present in the coding sequence of BhAPRR2 in green-skinned wax gourd were absent from white-skinned wax gourd. The latter contained a frameshift mutation, a premature stop codon, and lacked 335 residues required for the protein functional region. The chlorophyll content and BhAPRR2 expression were significantly higher in green-skinned than in white-skinned wax gourd. Thus, BhAPRR2 may regulate the peel color of wax gourd. This study provides a theoretical foundation for further studies of the mechanism of gene regulation for the fruit peel color of wax gourd.

Fine Mapping and Identification of the Candidate Gene BFS for Fruit Shape in Wax Gourd (Benincasa Hispida)

Fruit shape is an important agronomic trait in wax gourds [Benincasa hispida (Thunb) Cogn.]. However, the candidate genes for this important trait, and their genetic mechanisms, remain unknown. In this study, we identified a candidate gene for fruit shape in wax gourds using a next-generation sequencing-based bulked segregant analysis in F2 populations derived from a cross between GX-71 (long cylindrical fruit, FSI = 4.56) and MY-1 (round fruit, FSI = 1.06) genotypes. According to bulked segregant analysis, the candidate gene is located in the 17.18 Mb region on chromosome 2, and the kompetitive allele specific polymerase chain reaction (KASP) marker was used to reduce it to the 19.6 Kb region. There is only one gene in the corresponding region of the reference genome, Bch02G016830 (designated BFS). We sequenced BFS in six wax gourd varieties with different fruit shapes. Sequence analysis showed that there were two non-synonymous mutations in the spherical wax gourd and one non-synonymous mutation in the cylindrical wax gourd. Quantitative real‑time polymerase chain reaction (qRT-PCR) analysis showed that the expression of BFS in round fruits was significantly higher than in long cylindrical fruits at the ovary formation stage. Therefore, BFS is a candidate gene for determination of the fruit shape of wax gourds. The predicted protein encoded by the BFS gene belongs to the IQD protein family, which have the structural characteristics of scaffold proteins and coordinate Ca2+ CaM signaling from the membrane to the nucleus. The BFS gene can assist with the breeding of new varieties that possess ideal fruit shapes.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Benincasa hispida in Korea

Benincasa hispida (Thunb.) Cogn. (syn. B. cerifera Savi, Cucurbita hispida Thunb.), called wax gourd or ash gourd, is a cucurbitaceous vine grown for medicinal purposes and commercial values of its large fruits in Southeast Asia (Al-Snafi 2013). During the summer and autumn of 2020, leaves of wax gourd were observed to be affected by powdery mildew with 100% disease incidence in an experimental plot of Jeonbuk National University (35°50′55″N, 127°07′48″E), Korea. Fungal colonies were initially circular to irregular, forming white patches on both sides of the leaves and young stems, finally covering entire leaves and causing premature senescence of the leaves and poor growth. A representative voucher specimen was deposited in the Korea University herbarium (KUS-F32171). At least 30 measurements were taken for each asexual diagnostic features. Conidiophores arising from superficial hyphae were straight, 100 to 210 μm long, and produced 3 to 7 immature conidia in chains with a crenate outline. Foot-cells were cylindrical, 46 to 74 ×10 to 12 μm, followed by 1 to 2 shorter cells. Conidia were ellipsoid-ovoid to barrel-shaped, 30 to 40 × 18 to 23 μm with a length/width ratio of 1.4 to 2.0 and contained conspicuous fibrosin bodies. Germ tubes were produced from a lateral position on conidia. Sexual stage was not observed during the growing season. The morphological characteristics of the fungus were compatible with those of Podosphaera xanthii (Castagne) U. Braun & Shishkoff (Braun and Cook 2012), a well-known cucurbitaceous powdery mildew. DNA was extracted from mycelium, and primer sets ITS1F/PM6 and PM3/TW14 were used for amplification of ITS1-5.8S-ITS2 regions and 5´-end of 28S rDNA gene, respectively (Takamatsu and Kano 2001). Sequences determined in this study were deposited to the GenBank under the accession numbers MW559231 and MW559420, respectively. The sequences for ITS regions and 28S rDNA gene showed 99.78% and 99.07% similarity respectively with those of P. xanthii (MH465242, MH465243, MT250855 for ITS, and MK357436, MT826247 for LSU). Pathogenicity was confirmed twice by pressing a diseased leaf onto young leaves of five wax gourd plants. Five non-inoculated plants were used as controls. Inoculated leaves developed symptoms after 5 days, whereas the control plants remained symptomless. The fungus present on the inoculated plants was identical morphologically to that originally observed on diseased plants. Sphaerotheca fuliginea (syn. P. xanthii) on B. hispida has been listed in Hungary, India, Japan, Singapore, and Taiwan so far (Farr and Rossman 2021). Recently, the identity of P. xanthii on B. hispida in Taiwan was confirmed with morphological examination and molecular analysis by Wu and Kirschner (2017). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on B. hispida in Korea. Since wax gourd production is only recently started on a commercial scale in the southern part of Korea, powdery mildew infections pose a serious threat to the safe production of the fruits, especially in organic farming where chemical control options are limited.