The fruit of cucumber (Cucumis sativus) is an important reproductive organ, whose quality of appearance and flavor directly affect whether consumers want to buy and eat it. In particular, the length of fruit is a critical agronomic trait that affects the yield and appearance quality, serving also as the main reference standard for the classification of fresh commercial fruit products (Qi et al., 2013). The fruit length of wild cucumber plants is only about 3-5 cm, whereas that of cultivated cucumber has changed significantly after its long-term domestication and improvement. Accordingly, fruit length is also a valuable trait targeted in current cucumber breeding efforts.

Before the entire cucumber genome was successfully sequenced, many researchers had studied the quantitative trait loci (QTLs) related to the shape and size of cucumber fruits, but almost no genes were cloned (Yuan et al., 2007; Yuan et al., 2008; Wang et al., 2009). At present, there are many reports on the genetic analysis, location, gene cloning and functional research of cucumber fruit’s shape, size, and length. Although many QTLs linked to the shape or size of cucumber fruit have been identified in recent years, very few of these QTLs have yet to be cloned. Qi et al. (2013) obtained five QTLs (fl1.1, fl3.1, fl4.1, fl4.2 and fl6.1) related to cucumber fruit length by analyzing the resequencing results of 115 cucumber materials. Later, Wei et al. (2014) used the inbred lines CC3 and NC76 that differ greatly in fruit length to construct an F₂ population, and then analyzed the length of commercial as well as mature fruit, which yielded eight QTLs relevant to fruit length. Weng et al. (2015) used different isolated populations to investigate fruits’ phenotype at different development stages, thereby obtaining 12 QTLs related to fruit size and shape. Zhu et al. (2016) found seven QTLs related to fruit length and eight QTLs related to fruit diameter by using a F₂ population. Among the above loci, the QTL located by different researchers but which overlap in the cucumber chromosome position is referred to as the consensus QTL. Weng et al. (2015) put forward a model to explain the differences in cucumber fruit shape based on 12 consistent QTLs—FS1.1, FS1.2, FS2.1, FS2.2, FS3.1, FS3.2, FS3.3, FS4.1, FS5.1, FS6.1, FS6.2, FS7.1—among them, FS1.1 plays a decisive role in fruit length, whereas only FS3.3 regulates fruit elongation. Yet the model can only explain about 40% of the fruit length variation effect, which suggests other genes contribute to controlling fruit size. In later studies, Sheng et al. (2019) and Pan et al. (2020) added another nine consistent QTLs (FS1.3, FS1.4, FS2.3, FS4.2, FS4.3, FS5.2, FS5.3, FS6.3, FS7.2) to the cucumber fruit’s shape regulation model ( Figure 1 ); among them, only FS4.3 regulates the fruit length of cucumber. Most of these 21 QTLs can be detected in many different populations of cucumber plants, which also shows that their regulation can cover most of the extant trait variation in the shape of cucumber fruit.

Although researchers have performed much QTL mapping and analysis of cucumber fruit shape, many QTLs have not been cloned. With more in-depth basic research, increasingly more reports are becoming available on the fine mapping, gene cloning, and molecular mechanism of cucumber fruit concerning its size and shape. For example, Pan et al. (2017) used different populations to locate candidate genes responsible for round cucumber via marker linkage analysis, these being homologous to tomato SlSUN genes. Wu et al. (2018) located the candidate gene of FS2.1 to be CsTRM5, which belongs to the homologous gene SlTRM5, a member of the tomato TONNEAU1 Recruiting Motif (TRM) family. In other work, by analyzing the sequence of 150 cucumber materials having different fruit lengths, Zhao et al. (2019) identified that the MADS-box family gene CsFUL1 regulates the length of cucumber fruit. Through a QTL-seq analysis, Xu et al. (2020) located the major gene CsFnl7.1 for cucumber neck length, which encodes a late embryogenesis abundant protein. More recently, using a NIL population, Che et al. (2023) identified the gene controlling the length of cucumber fruit as CsRCR, and analyzed the regulation mechanism of its alleles in the formation of cucumber fruit length.

By studying mutants of cucumber fruit length, researchers have also uncovered some quality trait genes that govern cucumber fruit length. For instance, Xin et al. (2019) obtained a short-fruit mutant short fruit1, generated by EMS (Ethylmethanesulfonate) mutation. Through BSA-seq and dCAPS marker screening, it was determined that SF1 is the key gene regulating cucumber fruit elongation by regulating ethylene. Zhang et al. (2020) used short fruit 2, a natural mutant of short-fruit cucumber, to determine that the gene SF2 regulates cucumber fruit length by regulating cell proliferation, which encodes an HDC1 homolog. Cheng et al. (2021) recently mapped a candidate gene to CsaV3_1G044310 by using a natural mutant msf (medium short-fruit), which encodes a homologous protein of the Arabidopsis type II inositol polyphosphate 5-phosphatase (type II 5 Ptase). Finally, Zhang et al. (2023) obtained a short-fruit mutant sf4 by EMS mutation. Using map-based cloning, the gene that controls cucumber fruit elongation was identified as Csa1G665390 on chr1, which encodes an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT).

In this study, RIL (recombinant inbred line) populations were used to construct a genetic linkage map containing 600 bin markers by resequencing, and the population’s genetic background was analyzed. Based on the phenotypic data from spring 2021 and autumn 2022, QTL analysis of major loci of cucumber fruit length was carried out. Three coincident QTLs located on Chr2, Chr4, and Chr6 were obtained in both seasons, among which Fl4.1 was the prominent interval, in that it contained 115 genes, of which 41 were significantly differentially expressed in the short-fruit line (S190) and long-fruit line (L67). Through gene function annotations we found that this interval contains a gene Csa4G337340 encoding an auxin efflux carrier family protein; the expression level of this gene in the long-fruit lines is obviously down-regulated. According to the transcriptomics analysis, numerous genes were enriched in the plant hormone signal transduction pathway, and many differentially expressed transcription factors were found. Therefore, we speculate that plant hormones and their related genes in addition to a large number of transcription factors together play a pivotal role in regulating the length of cucumber fruit.

In this study, we analyzed the major QTLs controlling cucumber fruit length, and then explored the key genes and pathways related to cucumber fruit length by conducting a transcriptome analysis. The two-season phenotypic investigation demonstrated that the fruit length of 151 RIL lines was basically the same, irrespective of season ( Figure 4 ), which suggests this trait is relatively stable in this RIL population. Among the QTLs distinguished in this study, the four QTLs of Fl2.1, Fl2.2, Fl4.1, and Fl6.1 in spring 2021, and the three QTLs of Fl2.1, Fl4.1, and Fl6.1 in autumn 2022, three were evident shared between seasons. Importantly, in both seasons the major QTL is Fl4.1, with LOD values of 7.82 and 6.71, respectively ( Figure 5 ; Table 2 ). This locus is reportedly related to fruit length, fruit shape, and fruit stalk length, as shown in many previous studies. Indeed, has been identified as one of the consistent QTLs affecting cucumber fruit shape during fruit development in studies by Weng et al. (2015) and Sheng et al. (2019).

Auxin is a fundamental endogenous hormone that participates in many vital processes of plant growth and development, such as embryogenesis, organogenesis, cell determination and division, and tropic responses (Enders and Strader, 2015). These processes are finely coordinated by auxin, namely by requiring the polar distribution of auxin within tissues and cells. The intercellular directionality of auxin flow is closely related to the asymmetric subcellular location of PIN auxin efflux transporters (Zhou and Luo, 2018). In our study, further analysis of those genes in the interval Fl4.1 shows that it contains 115 genes. According to their gene function annotations, the Csa4G337340 gene in this region encodes an auxin efflux carrier family protein. According to this gene’s expression levels and patterning in different tissues of cucumber, as provided by the cucumber genome website, we find it maximally expressed in the unfertilized ovary ( Figure 6A ). From the RT-PCR, it is clear that expression levels of this gene are significantly lower in the long-fruit parent XTMC and the longest fruit RIL lines L67, L35, and L58 than those in the short-fruit parent HW and the shortest fruit RIL lines S190, S44-2, and S126 ( Figure 6B ). Combined with previous research findings, these results led us to speculate that Csa4G337340 may affect the content of auxin in cucumber fruit by participating in its auxin transport, and thereby play a negative regulatory role in determining cucumber fruit length.

Plant endogenous hormones are crucial for normal fruit development, because they can individually or interactively influence plants’ tissue or organ development (Vogel, 2006; Su et al., 2011; Meng X. et al., 2015; Ma and Li, 2019). Notably, Zhang (2019) used the short fruit mutant sf2 and wild-type cucumber to analyze the transcriptomics of their fruits at different developmental stages, finding that SF2 regulated the cell division of fruits through the synergistic regulation of various hormone pathways, such as IAA, GA, CK, ABA, JA and ETH, which then affected the fruit cell division. Recently, Hu et al. (2022) examined the hormones and hormone-related genes of the wild type and long-fruited mutant lf, showing that, compared with the wild type, the lf mutant’s fruit content of cytokinin and auxin changed significantly, as well as the expression of related genes; this indicates that auxin and cytokinin may be involved in the elongation of cucumber fruit cells. In the present study, from the transcriptomic analysis of fruits produced by shortest fruit line S190 and the longest fruit line L67 of the RIL populations, numerous DEGs were enriched in ‘plant hormone signal transduction’ pathway. Those enriched in auxin signal transduction pathway are the most abundant, totaling 67 DEGS, with 25 up-regulated and 42 down-regulated; another 27 DEGs were enriched in cytokinin signal transduction pathway (9 up-regulated, 18 down-regulated); In addition, a large number of genes are enriched in signal transduction pathways such as ethylene, gibberellin, abscisic acid, salicylic acid, and jasmonic acid. Therefore, we speculate that cucumber fruit length may be related to the content of hormones and the expression of hormone-related genes in fruit, a view consistent with other reported findings (Matsuo et al., 2012; Zhao et al., 2019).

Finally, we also found 509 differentially expressed transcription factors between S190 and L67, most of which belong to the BHLH, AP2/ERF-ERF, C2H2, NAC, Bzip, and MYB gene families. Many of them function in mediating cell division and expansion ( Figure 12 ). In work by Jiang et al. (2015), a large number of transcription factors were found that could be related to cucumber fruit development, mainly concentrated in the MYB, BHLH, NAC, and ERF/AP2 gene families, including some reported transcription factors such as SPT, IND, CRC, FUL, SUP, and HAN (Heisler et al., 2001; Girin et al., 2011; Zhang et al., 2013). Hence, we speculate that transcription factors also figure prominently in regulating the determination of cucumber fruit length.

A RIL population with 151 lines were derived from the cross between ‘HW’ and ‘XTMC’ and a high-density genetic map was constructed. Combined with the phenotypic data of different seasons, the QTL controlling the fruit length was located, and the major QTL was Fl4.1, which contained 115 genes. Based on the gene function annotations, we found a gene Csa4G337340 encoding an auxin efflux carrier family protein, whose expression level in the long-fruit parent and three longest-fruit RIL lines is obviously down-regulated. Transcriptome sequencing was carried out on the fruits of shortest-fruit line S190 and longest-fruit line L67, and KEGG enrichment results showed that differentially expressed genes were significantly enriched in the plant hormone signal transduction pathway. In addition, 509 transcription factors were detected among the DEGs, these distributed in various transcription factor gene families, and many of them were reported to be involved in the regulation of phytohormone synthesis and metabolism. Accordingly, we conclude that the major gene controlling the fruit length of cucumber is located in the interval of Fl4.1, and the gene Csa4G337340 in this interval may be involved in the negative regulation of fruit length. In addition, genes related to plant hormone signal transduction and several transcription factors are also involved in the regulation of cucumber fruit length.

The data presented in the study are deposited in the NCBI database repository, accession number PRJNA1005009.

YX, YC and YM were mainly responsible for the investigation and statistics of phenotypic data. YX wrote the first draft and analyzed the transcriptomic data. FW and SX were responsible for the cultivation and management of cucumber materials. WZ conceived the idea and corrected the paper to present form. All authors read and approve the same for publication.