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Introduction: Chieh-qua ( Benincasa hispida Cogn. var. Chieh-qua How) is a wax gourd variety that is generally susceptible to infection and damage by Fusarium oxysporum during its cultivation. Therefore, analyzing the adaption mechanism of chieh-qua to F. Oxysporum infection is of great significance for cultivating resistant varieties., Methods: Through comparative transcriptome analysis, comparative metabolome analysis, integrated analysis of transcriptome and metabolome and between F. Oxysporum infected samples and control samples of susceptible lines., Results: This study found that proteins such as NPR1, TGA and PR1 in plant hormone signal transduction pathway were up-regulated after infection, which may activate a series of plant secondary metabolic synthesis pathways. In addition, the expression of 27 genes in the flavonoid biosynthetic process in resistant lines after infection was significantly higher than that in susceptible lines, indicating that these genes may be involved in fungal resistance. This study also found that alternative splicing of genes may play an important role in responding to F. Oxysporum infection. For example, plant protein kinase genes such as EDR1, SRK2E and KIPK1 were not differentially expressed after F. Oxysporum infection, but the transcripts they produced differ at the transcription level. Finally, through comparative metabolome analysis, this study identified potentially functional substances such as oxalic acid that increased in content after F. Oxysporum infection. Through integrated analysis of transcriptome and metabolome, some differential expressed genes significantly related to differential metabolites were also identified., Discussion: This study provides a basis for understanding and utilizing chieh-qua's infection mechanism of F. Oxysporum through analysis of the transcriptome and metabolome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Qiao, Peng, Wu, Wang, He, Peng, Gao, Liu, Yang and Dai.)

Introduction: Flesh color is an essential trait in chieh-qua ( Benincasa hispida Cogn. var. Chieh-qua How); however, the inheritance and molecular basis of green flesh trait remain unclear., Methods: In the present study, two F 2 populations, derived from 1742 (white flesh) × FJ3211 (green flesh) and J16 (white flesh) × FJ5 (green flesh), were used to identify the green flesh ( Cqgf ) locus., Results: Genetic analysis revealed that the presence of green flesh was a quantitative trait that closely followed a normal distribution. Combining the results from QTL mapping and BSA-seq analysis, the Cqgf locus was preliminarily determined to be located on chromosome 05 and was narrowed down to a 2.55-Mb interval by linkage analysis. A large J16 × FJ5 F 2 population comprising 3,180 individuals was subsequently used to screen the recombinants, and the Cqgf locus was fine-mapped to a region of 329.70 kb that harbors six genes. One of the candidate genes, Bch05G003700 , the zinc-finger transcription factor LOL1 (lsd one like 1 protein; CqLOL1 ), was the strongest candidate gene for the Cqgf locus according to sequence variation and expression analysis. Additionally, a point mutation (A > C) in CqLOL1 resulted in the substitution of threonine (T) with proline (P) in the amino acid sequence, showing a complete relationship linked with flesh color in a panel of 45 germplasms., Discussion: The study suggests that CqLOL1 promotes the accumulation of chlorophyll content in chieh-qua and lead to green flesh. Our findings establish a theoretical and technical foundation for breeding different flesh color lines and elucidating the underlying mechanisms of flesh color in chieh-qua., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Peng, Gao, Qiao and Wang.)

Chieh-qua (Benincasa hispida Cogn. var. Chieh-qua How.) fruit development starts post pollination. With the continuous expansion of the fruit, the soluble solid content of the fruit decreases. Because there are no reports on the early development of Chieh-qua fruit, this study compared fruit transcriptomes at 0-, 3-, and 7 day post pollination (dpp). 104,747 unigenes were assembled from clean reads and compared using six public databases for similarity searching. Compared with those of 0 dpp (C), there were differences in the expression of 12,982 and 6541 genes in the fruit tissue at 3 dpp and 7 dpp, respectively. Compared with 3 dpp (B), there were 14,314 differentially expressed genes in the fruit at 7 dpp (A). Based on the analysis of transcription factors, 213 nucleotides in the MYB superfamily were identified; among them, 94 unigenes of the MYB superfamily were differentially expressed at the three stages. In the pairwise comparison of differential expression, eight unigenes (Gene_id: TRINITY_DN32880_c1_g2, TRINITY_DN35142_c2_g2, TRINITY_DN32454_c11_g6, TRINITY_DN34105_c2_g7, TRINITY_DN32758_c3_g3, TRINITY_DN33604_c4_g10, TRINITY_DN34466_c3_g1, TRINITY_DN35924_c3_g2) were homologous to those of MYB59, MYB-GT3b, MYB18, MYB4, MYB108, MYB306, MYB340, and MYB-bHLH13. These unigenes differed significantly among the three stages. Furthermore, MYB59 and MYB18 exhibited higher expression at 7 dpp. MYB4, MYB-GT3b, MYB108, and MYB306 showed the highest expression levels in fruits at 3 dpp. In addition, MYB340 and MYB-bHLH13 showed higher expression levels during the unpollinated stage. MYB59, MYB-GT3b, MYB18, MYB4, MYB108, MYB306, MYB340, and MYB-bHLH13 may play crucial roles in Chieh-qua fruit development, defense, and blossoming. This study provides a basis for further investigation of MYB superfamily genes involved in early fruit expansion in chieh-qua., (© 2024. The Author(s).)

Seeds are the most important reproductive organs of higher plants, the beginning and end of a plant's lifecycle. They are very important to plant growth and development, and also an important factor affecting yield. In this study, genetic analysis and BSA-seq of the F 2 population crossed with the large-seeded material 'J16' and small-seeded material 'FJ5' were carried out, and the seed size locus was initially located within the 1.31 Mb region on chr10. In addition, 2281 F 2 plants were used to further reduce the candidate interval to 48.8 Kb. This region contains only one gene encoding the N-acetyltransferase (NAT) protein ( Bch10G006400 ). Transcriptome and expression analysis revealed that the gene was significantly more highly expressed in 'J16' than in 'FJ5'. Variation analysis of Bch10G006400 among parents and 50 chieh-qua germplasms revealed that as well as a nonsynonymous mutation (SNP_314) between parents, two mutations (SNP_400 and InDel_551) were detected in other materials. Combining these three mutations completely distinguished the seed size of the chieh-qua. GO and KEGG enrichment analyses revealed that DGEs played the most important roles in carbohydrate metabolism and plant hormone signal transduction, respectively. The results of this study provide important information for molecular marker-assisted breeding and help to reveal the molecular mechanism of seed size.

The cucurbit vegetable chieh-qua ( Benincasa hispida var. chieh-qua How) is an important crop in South China and southeast Asian countries. Viral diseases cause substantial loss of chieh-qua yield. To identify the viruses that affect chieh-qua in China, ribosomal RNA-depleted total RNA sequencing was performed using chieh-qua leaf samples with typical viral symptoms. The virome of chieh-qua comprises four known viruses (melon yellow spot virus (MYSV), cucurbit chlorotic yellows virus (CCYV), papaya ringspot virus (PRSV) and watermelon silver mottle virus (WSMoV) and two novel viruses: cucurbit chlorotic virus (CuCV) in the genus Crinivirus and chieh-qua endornavirus (CqEV) in the genus Alphaendornavirus . The complete genomes of the two novel viruses in chieh-qua and three other isolates of CuCV in pumpkin, watermelon and cucumber were determined and the recombination signals of pumpkin and watermelon isolates of CuCV were detected. A reverse transcriptase PCR indicated that the dominant viruses of chieh-qua in Hainan are MYSV (66.67%) and CCYV (55.56%), followed by CuCV (27.41%), WSMoV (7.41%), cucumber mosaic virus (8.15%), zucchini yellow mosaic virus (6.67%), PRSV (6.67%) and CqEV (35.56%). Our findings support diagnostic and prevalence studies of viruses infecting chieh-qua in China, enabling sustainable control strategies for cucurbit viruses worldwide.

Gynoecy demonstrates an earlier production of hybrids and a higher yield and improves the efficiency of hybrid seed production. Therefore, the utilization of gynoecy is beneficial for the genetic breeding of chieh-qua. However, little knowledge of gynoecious-related genes in chieh-qua has been reported until now. Here, we used an F 2 population from the cross between the gynoecious line 'A36' and the monoecious line 'SX' for genetic mapping and revealed that chieh-qua gynoecy was regulated by a single recessive gene. We fine-mapped it into a 530-kb region flanked by the markers Indel-3 and KASP145 on Chr.8, which harbors eight candidate genes. One of the candidate genes, Bhi08G000345 , encoding networked protein 4 ( CqNET4 ), contained a non-synonymous SNP resulting in the amino acid substitution of isoleucine (ATA; I) to methionine (ATG; M). CqNET4 was prominently expressed in the female flower, and only three genes related to ethylene synthesis were significantly expressed between 'A36' and 'SX.' The results presented here provide support for the CqNET4 as the most likely candidate gene for chieh-qua gynoecy, which differed from the reported gynoecious genes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wang, Yang, Liu, Cao, Chen, Liu, Xie, Yan, Jiang and Peng.)

Temperature rising caused by global warming has imposed significant negative effects on crop qualities and yields. To get the well-known molecular mechanism upon the higher temperature, we carefully analyzed the RNA sequencing-based transcriptomic responses of two contrasting chieh-qua genotypes: A39 (heat-tolerant) and H5 (heat-sensitive). In this study, twelve cDNA libraries generated from A39 and H5 were performed with a transcriptome assay under normal and heat stress conditions, respectively. A total of 8705 differentially expressed genes (DEGs) were detected under normal conditions (3676 up-regulated and 5029 down-regulated) and 1505 genes under heat stress (914 up-regulated and 591 down-regulated), respectively. A significant positive correlation between RNA-Seq data and qRT-PCR results was identified. DEGs related to heat shock proteins (HSPs), ubiquitin-protein ligase, transcriptional factors, and pentatricopeptide repeat-containing proteins were significantly changed after heat stress. Several genes, which encoded HSPs ( CL2311.Contig3 and CL6612.Contig2 ), cytochrome P450 ( CL4517.Contig4 and CL683.Contig7 ), and bHLH TFs ( CL914.Contig2 and CL8321.Contig1 ) were specifically induced after four days of heat stress. DEGs detected in our study between these two contrasting cultivars would provide a novel basis for isolating useful candidate genes of heat stress responses in chieh-qua.

The WRKY transcription factors play an important role in plant resistance for biotic and abiotic stresses. In the present study, we cloned 10 WRKY gene homologs (CqWRKY) in Chieh-qua (Benincasa hispida Cogn. var. Chieh-qua) using the rapid-amplification of cDNA ends (RACE) or homology-based cloning methods. We characterized the structure of these CqWRKY genes. Phylogenetic analysis of these sequences with cucumber homologs suggested possible structural conservation of these genes among cucurbit crops. We examined the expression levels of these genes in response to fusaric acid (FA) treatment between resistant and susceptible Chieh-qua lines with quantitative real-time PCR. All genes could be upregulated upon FA treatment, but four CqWRKY genes exhibited differential expression between resistant and susceptible lines before and after FA application. CqWRKY31 seemed to be a positive regulator while CqWRKY1, CqWRKY23 and CqWRKY53 were negative regulators of fusaric resistance. This is the first report of characterization of WRKY family genes in Chieh-qua. The results may also be useful in breeding Chieh-qua for Fusarium wilt resistance.

A simple and rapid method based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed for the simultaneous determination of imidacloprid and chlorfenapyr residues in chieh-qua. Field trials were designed to investigate the dissipation and terminal residue behavior of the mixed formulation of imidacloprid and chlorfenapyr in chieh-qua in Guangzhou and Nanning areas. Risk assessment was performed by calculating the risk quotient (RQ) values. The developed analytical method exhibited recoveries of 89.9-110.3% with relative standard deviations (RSDs) of 2.8-12.5% at the spiked levels of 0.01, 0.10, and 1.00 mg/kg. The limit of detection (LOD) was 0.003 mg/kg, and the limit of quantification (LOQ) was 0.01 mg/kg for both imidacloprid and chlorfenapyr. It was found that the half-lives of imidacloprid in chieh-qua under field conditions were 3.3 and 3.5 days in Guangzhou and Nanning at a dose of 180 g ai/ha, while the half-lives of chlorfenapyr were 3.3 and 2.6 days, respectively. The terminal residues of imidacloprid and chlorfenapyr were from 0.01 to 0.21 mg/kg and from 0.01 to 0.46 mg/kg, respectively. Results of dietary exposure assessment showed that the RQ values were much lower than 1, indicating that the risk of imidacloprid and chlorfenapyr applied in chieh-qua was negligible to human health under recommended dosage and good agricultural practices. The proposed study would provide guidance for safe and reasonable use of imidacloprid and chlorfenapyr in chieh-qua cultivation in China.