Cucurbita pepo L. is one of the cultivated Cucurbita spp. and has the most abundant germplasm resources among Cucurbita crops. Zucchini not only shows strong stress resistance and adaptability, but also had high nutritional, medicinal, and ornamental value. Therefore, zucchini is grown widely from tropical to temperate areas. The surfaces of the aboveground tissues of most zucchini germplasm resources are covered with sharp dense trichomes. During the field production of zucchini, the sharp dense trichomes of zucchini plants can cause skin itchiness in people managing or pollinating plants or harvesting their fruit. Furthermore, the dense trichomes of fruit stems easily scratch young zucchini fruits, which is unfavorable for fruit transport. Therefore, it is necessary to identify and clone genes controlling trichome density trait in zucchini.

Trichomes are the first functional barrier in contact with the outside world and play a significant role in plant development and the biotic and abiotic adaptations, and some of them have important commercial value (Amme et al., 2005; Emiko et al., 2010; An et al., 2012). Trichomes are widely distributed on the surfaces of the aboveground parts of land plants, such as leaves, petioles, stems, pericarps, fruits, tendrils, and pedicels (Liu et al., 2016). Different arrangements and numbers of trichome cells develop into different morphologies, and according to trichome morphology, plant trichomes are divided into unicellular or multicellular trichomes (Schnittger et al., 1999). Unicellular trichomes are nonglandular, and some have branches, while multicellular trichomes are divided into glandular or nonglandular trichomes (Schnittger et al., 1999). Common multicellular trichomes had complex structures and consist of three components: head cells, stalk cells, and basal cells (Amme et al., 2005; Chang et al., 2016).

Trichomes in Arabidopsis are typically unicellular branched structures. Numerous genes have been identified as key regulators of trichome initiation. The regulatory network affecting trichome initiation and development was constructed, and approximately 40 genes were involved. In Arabidopsis, the R2R3 MYB transcription factor GLABRA 1 (GL1) (Kirik et al., 2004), the basic helix-loop-helix (bHLH) transcription factor GLABRA 3/ENHANCER OF GLABRA 3 (GL3/EGL3), and the pleiotropic WD40 repeat protein TRANSPARENT TESTA GLABRA 1 (TTG1) (Walker et al., 1999; Zhao et al., 2008) formed the MYB-bHLH-WDR (MBW) complex, which was the key core of the trichome initiation pathway. The MBW complex activates trichome formation by enhancing the expression of downstream homeodomain-leucine zipper (HD-ZIP) IV genes, GL2 and EGL2 (Payne et al., 2000; Ishida et al., 2007). R3-type MYB transcription factors CAPRICE (CPC), ENHANCER OF TRY AND CPC 1 (ETC1), ETC2, ETC3, TRIPTYCHON (TRY), TRICHOMELESS 1 (TCL1), and TCL2 act as negative regulators of trichome formation by competing with GL1 and interacting with GL3/EGL3 to form an inactive complex (Schnittger et al., 1999; Kirik et al., 2004; Wang et al., 2007; Wang et al., 2008; Wester et al., 2009). bHLH transcription factor MYC1 positively regulates trichome number (Zhao et al., 2012). C2H2 type zinc-finger proteins (ZFPs), such as glabrous inflorescence stems (GIS), GIS2, GIS3, ZFP5, ZFP6, and ZFP8, play key roles in the initiation and formation of leaf trichomes (Gan et al., 2007; Zhou et al., 2012; Zhou et al., 2013; Sun et al., 2015; Liu et al., 2017; Kim et al., 2018). The AP2/ERF family transcription factor TARGET OF EARLY ACTIVATION TAGGED 1 (TOE1) regulates trichome initiation of the main-stem inflorescence (Liu et al., 2023). SET Domain Group 26 (SDG26), a histone lysine methyltransferase, affects trichome growth and development (Zeng et al., 2023).

Tomato trichomes have unbranched multicellular structures and are divided into eight types. Types I, IV, VI, and VII are glandular types with secretory capacity, whereas types II, III, V, and VIII are nonglandular types (Amme et al., 2005). The functions of an HD-ZIP IV family gene, Woolly (Wo), are involved in the formation and development of type I and IV trichomes (Yang et al., 2011). A B-type cyclin gene, SlCycB2, participates in the development of type I, III, and V trichomes (Gao et al., 2017). SlMYC1, a bHLH transcription factor, regulates not only the development of type VI trichomes but also mono- and sesquiterpene biosynthesis in tomato (Xu et al., 2018). An R2R3-MYB family gene, SlMIXTA, functions as a positive regulator of tomato trichome formation (Ying et al., 2020). SlHair (SlH), a C2H2 zinc finger protein (ZFP), positively regulates the number and length of type I trichomes in tomato (Chun et al., 2021).

Cucumber fruit is covered with spines (a special type of trichome) and tubercules, which are important agronomic traits that affect commercial value. Fruit spines have branchless multicellular structures. Fruit spines are divided into eight types: types I and VI are glandular and types II, III, IV, V, VII, and VIII are nonglandular (Xue et al., 2019). The HD-ZIP I transcription factor genes Glabrous 1/Tny branched hair/Micro-trichome (CsGL1/TBH/MICT) are key allelic genes involved in spine development and affect spine distribution in cucumber (Chen et al., 2014; Li et al., 2015; Zhao et al., 2015). The HD-ZIP IV transcription factor genes Glabrous 3/Trichome-less/Few spine 1 (CsGL3/TRIL/FS1) are key allelic genes for the positive regulation of cucumber fruit spines’ differentiation, initiation, and development (Liu et al., 2016; Wang et al., 2016; Zhang et al., 2016; Zhang et al., 2019). The C2H2-type zinc finger gene tuberculate (Tu) is a key gene that controls the formation of tuberculate fruit (Yang et al., 2014). The WD-repeat homologues CsTTG1 and MIXTA-LIKE transcription factor CsMYB6 are key factors involved in regulating the differentiation of fruit spines and the tubercule formation (Chen et al., 2014; Chen et al., 2016; Zhao et al., 2020). bHLH transcription factors, such as CsMYC2, CsMYC4, CsMYC5, CsMYC6, CsMYC7, and CsMYC8, play an important role in glandular trichome development (Feng et al., 2023). In addition to transcription factor genes, other genes are also involved in cucumber spine development. NUMEROUS SPINES (NS) acts as a regulatory factor in spine development by modulating the auxin signaling pathway (Xie et al., 2018). The tender spines (TS) gene encodes a C-type lectin receptor-like tyrosine-protein kinase and plays an important role in the formation of cucumber spines (Guo et al., 2018).

Gene mapping studies and analyses of regulatory mechanisms related to trichome/spine initiation and development are mainly conducted in model plants, such as Arabidopsis, tomato, and cucumber. In a previous report, zucchini trichomes were classified into seven types according to the morphology: type I, II, III, and VII trichomes were nonglandular, and type IV, V, and VI trichomes were glandular. Among them, type I and II trichomes were the longest. The zucchini germplasm resources were divided into a long dense trichome (LDT) group and a sparse micro trichome (SMT) group according to the presence of type I and II trichomes. An F₂ population derived by crossing the LDT line “16” and SMT line “63” was constructed, and the trichome density exhibited characteristic quantitative inheritance (Lin et al., 2023). Although there have been reports of trichome characteristics, classification, and inheritance, the key genes and regulatory mechanisms related to zucchini trichome development remain to be reported. In our study, quantitative trait loci (QTLs) related to the trichome density were identified using the QTL sequencing (QTL-seq) method and genetic map-based QTL analysis. Additional F₂ populations grown under different environmental conditions were used to validate the stability of QTLs, and the candidate genes in the major-effect QTL region associated with trichome development were identified. A C3DHC3-type ZFP gene might regulate trichome density in zucchini. The results lay a foundation for better understanding of multicellular trichome development and improving trichome-related breeding in zucchini.

Trichomes are hairy structures covering aboveground tissues in terrestrial plants and provide an excellent model for studying cell differentiation and proliferation. Trichome development involves trichome density, trichome number, trichome distribution, trichome morphology, the production of secondary metabolic secretions, and so on. The R2R3 MYB, bHLH, HD-ZIP, and C2H2 ZFP families of transcription factors play significant roles in the regulation of trichome development in the model plants Arabidopsis thaliana, tomato, and cucumber. There are few reports on regulatory genes and trichome development mechanisms in zucchini. In our previous study, the multicellular trichome morphology of aboveground tissues of “16” and “63” was observed, and trichomes were classified into seven types: type I, II, III, and VII trichomes were nonglandular, and type IV, V, and VI trichomes were glandular. Type I and II trichomes were longer and harder than the other types, and the presence or absence of type I and II trichomes was an obvious difference between “16” and “63” (Lin et al., 2023). Type I and II trichomes of petioles were larger than those on other tissues and were dispersedly distributed on the surface; therefore, we could more easily observe trichome morphology and density by microscopy. As demonstrated by observing the density of type I and type II trichomes on petioles, the phenotypes of F₂ plants were categorized into five classes. F₂ individuals of class 1 had the sparsest type I and type II trichomes, and individuals of the class 5 phenotype had the densest type I and type II trichomes (Lin et al., 2023). In cucumber, the developmental processes involved in glandular and nonglandular multicellular trichome formation were observed using cotyledons as a model and divided into five sequential stages (Dong et al., 2022). In our study, zucchini cotyledons were used as a model and the same developmental processes of glandular and nonglandular trichomes were observed. The third stage (tip head formation/glandular head transition) was the key stage for glandular and nonglandular trichome development. The morphological observation and classification of zucchini trichomes laid a foundation for the QTL analysis of trichome density.

In our study, an F₂ population derived by crossing “16” and “63” was constructed, and QTL analysis of the density of type I and type II trichomes was performed with QTL-seq and genetic map-based QTL analysis. Two QTLs associated with trichome density, CpTD3.1 and CpTD15.1, were detected on chromosomes 3 and 15 by both QTL analysis methods. The stability of CpTD3.1 and CpTD15.1 was confirmed using data from three environmental conditions. The results showed that CpTD3.1 and CpTD15.1 were detected in similar regions in all environments, which explained 5%–10.64% and 12.71%–29.37% of the observed phenotypic variation, respectively. These two QTLs showed good stability, and the environment seldom affected the major-effect QTL CpTD15.1 or the minor-effect QTL CpTD3.1. The CpTD3.1 locus was within a 2,672.89-kb region between markers chr3-218350 and chr3-2891236, and the CpTD15.1 locus was within a 775.44-kb region between markers chr15-4991349 and chr15-5766791. The genetic interaction analysis indicated that there was a simple additive gene effect between CpTD3.1 and CpTD15.1. F₂ individuals with the CpTD15.1 locus derived from parent “16” and the CpTD3.1 locus derived from parent “63” were labeled 15A-3B, individuals with the CpTD15.1 locus derived from parent “63” and the CpTD3.1 locus derived from parent “16” were labeled 15B-3A, and individuals with the CpTD15.1 and CpTD3.1 loci derived from parent “16” were labeled 15A-3A. The phenotypes of 15A-3B, 15B-3A, and 15A-3A in 2020F were collected. The trichome density of 15A-3B ranged from class 2 to class 4 with an average class of 2.86, the trichome density of 15B-3A ranged from class 1 to class 3 with an average class of 2.04, and the trichome density of 15A-3A ranged from 3 to 5 with an average class of 3.74 ( Figure S3 ). The results confirmed that zucchini plants with both loci derived from parent “16” had dense trichomes. Conventional breeding techniques require the selection of phenotypes and evaluation in multiple environments for many years, and this process could require considerable time, labor, and land. Marker-assisted selection (MAS) is a powerful tool for selecting the target trait, which effectively accelerates the breeding process. Markers closely linked to the CpTD15.1 and CpTD3.1 loci (chr3-218350, chr3-2891236, chr15-4991349, and chr15-5766791) could potentially aid in the selection of trichome density at the seedling stage and be successfully used in MAS.

ZFPs are one of the most important transcription factors in plants. Based on the different number and arrangement of Cys and His residues, ZFPs can be categorized into nine types, namely, C2H2, C8, C6, C3HC4, C2HC, C2HC5, C4, C4HC3, and CCCH (Berg and Shi, 1996). ZFPs play vital roles in the regulation of growth, developmental processes, and multiple stress responses in plants (Bernhardt et al., 2003; Davletova et al., 2005; Dinneny et al., 2006; Xiao et al., 2009; Zhang et al., 2012). Many C2H2 ZFP genes have also been reported to be involved in trichome formation in unicellular or multicellular trichome plants (Davletova et al., 2005; Dinneny et al., 2006; Xiao et al., 2009; Zhang et al., 2012). In Arabidopsis, the first-identified C2H2 ZFP, GLABROUS INFLORESCENCE STEMS (GIS), induces trichome initiation in inflorescence organs. It acts upstream of the MYB-bHLH-WD complex and GL1 by gibberellin (GA), and acts downstream of SPINDLY to promote inflorescence trichome initiation (Gan et al., 2006). Two C2H2 ZFPs, GIS2 and ZFP8, are integrated with cytokinin and GA signaling in the regulation of trichome initiation on inflorescence organs and cauline leaves, respectively (Gan et al., 2007). ZFP5, ZFP6, and GIS3 are the key proteins involved in trichome initiation on inflorescence organs, acting upstream of GIS, GIS2, and ZFP8 (Zhou et al., 2012). In tomato, the SlH, SlHair2/SlZFP8L, and SlZFP6 genes are members of the C2H2 family. SlH increases the length of type I, III, and VI trichomes. SlH and its closest homologue, SlZFP8L, physically interact with Wo, and SlH, zinc finger protein-like 8 (SlZFP8L), and Wo function together to regulate long-stalk type I trichome initiation (Chang et al., 2018; Chun et al., 2021). SlZFP6 and SlZFP8L are critical for the density of type I, III/V, VI, and VII trichomes and increase the length of long-stalk type I, III, and VI trichomes. SlH interacts with SlZFP8-like to regulate trichome initiation and elongation by modulating SlZFP6 gene expression. Based on transgenic technology, SlH, SlZFP8L, and SlZFP6 may promote trichome elongation by activating the expression of cell-wall-loosening protein genes (Zheng et al., 2022). In conclusion, C2H2 zinc finger transcription factors play similar roles in the regulation of trichome formation in unicellular trichome plant Arabidopsis and multicellular trichome plant tomato. Cucumber fruits are covered with tubercules and spines. The tuberculate fruit gene Tu, which was identified as a C2H2 ZFP gene, is involved in the CTK biosynthetic pathway (Yang et al., 2014). In our research, genes encoding transcription factors were screened in the region of the major-effect locus CpTD15.1. Among these genes, one (Cp4.1LG15g04400) encoding ZFP harbored nonsynonymous SNPs in the conserved ring finger domain. The ring finger domain of Cp4.1LG15g04400 was of the C3DHC3 type, which is found in ZFPL1 genes. There were significant differences in Cp4.1LG15g04400 expression between the LDT parent “16” and the SMT parent “63”, and a similar pattern was shown between zucchini LDT lines and SMT lines. A phylogenetic analysis of Cp4.1LG15g04400 with known genes was performed, and Cp4.1LG15g04400 was found to be a distant relative of C2H2 ZFP genes ( Figure S4 ). Based on sequence analysis, gene function annotation, and gene expression analysis, it was presumed that Cp4.1LG15g04400 might regulate trichome density as a new type of ZFP gene in zucchini. Near-isogenic lines of CpTD15.1 were constructed to confirm the candidate gene of the major locus. The gene functions and regulatory pathways related to the trichome density of Cp4.1LG15g04400 need to be further validated based on more molecular biological experiments in zucchini.

An F₂ population was constructed with the LDT inbred line “16” and the SMT inbred line “63” for QTL analysis of type I and II trichome density. Two QTLs, CpTD3.1 and CpTD15.1, were identified on chromosomes 3 and 15 using the QTL-seq method and genetic map-based QTL analysis, and CpTD15.1 was the major-effect QTL. The stability of CpTD3.1 and CpTD15.1 was confirmed using data from F₂ plants under different environmental conditions. The major-effect QTL CpTD15.1 was located between markers chr15-4991349 and chr15-5766791 with a physical distance of 775.44 kb, and explained 12.71%–29.37% of the phenotypic variation observed in the three environments. CpTD3.1 was located between markers chr3-218350 and chr3-2891236, with a physical distance of 2,672.89 kb, and explained 5.00%–10.64% of the phenotypic variation in the three environments. In the CpTD15.1 region, five genes encoding transcription factors regulating trichome development were selected. Cp4.1LG15g04400 harbored nonsynonymous SNPs in the conserved ring finger domain between the two parental lines. There were significant differences in Cp4.1LG15g04400 expression between “16” and “63” and in the germplasm resources of LDT lines and SMT lines. It was inferred that Cp4.1LG15g04400 might regulate trichome density in zucchini. This study lays a foundation for better understanding the density of multicellular nonglandular trichomes and the regulatory mechanism of trichome density in zucchini.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: BioProject accession number: PRJNA987358.

YW performed data analysis and prepared the manuscript. GW contributed to RNA extraction and qRT-PCR test. DL contributed to collecting phenotypic characteristics and DNA extraction. QL contributed to growing plants. WX and SQ, the corresponding authors, oversaw all activities related to the project implementation and manuscript development. All authors read and approved the final version of the manuscript.