Fine mapping of BhDDL4.1, a major gene controlling the regulation of the deeply lobed leaf trait in wax gourd (Benincasa hispida)

Fine mapping of BhDDL4.1, a major gene controlling the regulation of the deeply lobed leaf trait in wax gourd (Benincasa hispida) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Fine mapping of BhDDL4.1, a major gene controlling the regulation of the deeply lobed leaf trait in wax gourd (Benincasa hispida) Wenhui Bai, Peng Wang, Yan Deng, Zhihao Chen, Liwen Su, Zhikui Cheng, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4085732/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Jul, 2024 Read the published version in Euphytica → Version 1 posted 9 You are reading this latest preprint version Abstract Lobed leaves play a vital role in high-density cultivation and breeding of wax gourd. Thus, determining the molecular mechanisms underlying the development of lobed leaves is important. To this end, in this study, we aimed to resequence 105 recombinant inbred lines, constructed using the parental lines, GX-7 and my-1, to elucidate the molecular mechanisms underlying leaf development in wax gourd ( Benincasa hispida ). Genes associated with lobed leaves in wax gourds were first evaluated via quantitative trait loci (QTL) mapping. Next, the F2 population was expanded to 2,000 plants for fine mapping and candidate gene analyses. Thus, the candidate area is reduced to 1.129 Mb, located between the markers InDel980 and InDel853. Functional analyses of candidate genes were performed using gene functional annotation, coding sequence analyses, and expression analyses. Among 48 genes in the candidate region, only Bch04G012650 (termed BhDDL4.1) showed differences in expression between two parents. Using sequence differences of previously screened candidate genes, an InDel marker (InDel623) was developed in BhDDL4.1 for molecular marker-assisted breeding of wax gourd, and the accuracy rate was 74.03%. Our results indicate that BhDDL4.1 may play a key role in regulation of the lobed leaf trait; thereby, we provided a theoretical basis for further exploration of the molecular mechanisms underlying the lobed leaf trait in wax gourds. Wax gourd Lobed leaf Fine mapping Cucurbitaceae Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Wax gourd ( Benincasa hispida (Thunb.) Cogn.) (2n = 2x = 24), also known as pillow melon, broad melon, or big gourd, belongs to the family Cucurbitaceae. It is an annual herb originating in southern China and eastern India. It has a wide planting range and a long planting history of cultivation. It is one of the most important vegetables transported from southern to northern China (Hu et al. 2022 ). The leaves are responsible for photosynthesis, nutrient distribution, water transport, and gas exchange and are, therefore, considered economically important nutritional organs. Phenotypic differences in leaf shape occur within and between species (Nicotra et al. 2011 ); (Andres et al. 2014 ); (Rodriguez et al. 2014 ); (Jiao et al. 2016 )). Leaf shape in wax gourd can either be non-cracked, serrated, or deeply lobed (Ni et al. 2015 ). Additionally, the leaf shape is affected by genetic factors as well as external conditions, such as temperature and light (Tsukaya 2005 ); (Walter and Schurr 2005 ). It may affect plant growth, development, structure, and function as well as the final quality of fruits (Heng et al. 2020 ); (Karamat et al. 2021 )). Generally, only the upper leaves of those densely planted plants with larger leaves are fully exposed to sunlight and effectively engage in photosynthesis. The leaves of plants with small leaf areas are not obstructed by adjacent leaves and are, therefore, able to fully absorb sunlight and effectively utilize light energy to improve photosynthetic efficiency. This reduces transpiration in plants (Yates et al. 2010 ); (Wilf 1997 ). Additionally, leaf shape is associated with plant stress resistance (Lopez-Iglesias et al. 2014 ). In practice, crops with deeply lobed leaves are more suitable for high-density cultivation (Tu et al. 2013 ). Therefore, studies focused on investigation of the regulatory genes linked to wax gourd-lobed leaves may positively impact the efforts aimed at improving germplasm resources, increasing yield, and enhancing economic benefits. In recent years, many genes and gene families associated with the development of leaf lobes in plants, such as Arabidopsis thaliana , have been discovered. The class I homeodomain leucine zipper transcription factor LMI1 plays an important role in the formation of serrated lobes in Arabidopsis (Saddic et al. 2006a ). The LMI1HD-ZIP transcription factor contributes to variation in deeply lobed leaves in various crops. Members of the HD-ZipI family participate in many biological processes, including the plant leaf growth and development, leaf polarity establishment, and light signal transduction (Merelo et al. 2016 ); (Zhou et al. 2019 ); (Whitewoods et al. 2020 ). A comparison of leaf morphology in A. thaliana and related plants has shown that leaflet development depends on the homology domain protein REDUCED COMPLEXITY (RCO) (Vlad et al. 2014 ). Furthermore, the HD-Zip transcription factor GhLMI1-D1b, which is a homolog of LMI1-like genes, was associated with different phenotypic traits in cotton leaf (Andres et al. 2017 ). Polymorphic LMI1-like genes regulate the growth and development of watermelon and rapeseed (Wang et al. 2021 ). Lobed leaves are modulated by the incompletely dominant gene BnLLA10 in rapeseed and the homologous gene LOBED-LEAF1 ( BnLL1) in cabbage (Ni et al. 2015 ); (Hu et al. 2018 ). At the same time, both of these genes can regulate the inheritance of deep lobed leaf in kale (Ni et al. 2017 ); (Hu et al. 2020 ). Feather leaf traits in ornamental cabbage are modulated by the semi-dominant gene BoFL , which encodes an alpha-1,2-glucosyltransferase in Arabidopsis (Feng et al. 2020 ). BrLMI1 is an incompletely dominant positive regulator of lobed leaf formation in non-heading cabbage (Li et al. 2023 ). As a meristem identity regulator, LMI1 plays an important role in regulating the growth and development of meristems, leaf, or bract (Saddic et al. 2006b ). In cotton, genes that regulate lobed leaves belong to LMI1/RCO and KNOXI (Chang et al. 2019 ). RCO regulates the activity of cytokinins, thereby affecting leaf shape parameters (Hajheidari et al. 2019 ), while KnoX1 and RCO synergistically regulate the plant leaflet growth (Wang et al. 2022 ). Genes that regulate lobed leaves have been evaluated in Cucurbitaceae. The deeply lobed leaves of Cucurbitaceae are regulated by a single incompletely dominant gene, CpDll , which encodes an HD-Zip I transcription factor; in particular, its high expression may be related to the formation of deeply lobed leaves (Bo et al. 2022b ). Moreover, the HD-Zip transcription factor plays a major role in the presence of lobed leaves in watermelon (Xu et al. 2023 ). A single dominant gene, ClLL1 , regulates the growth and development of leaf lobes in watermelon (Wei et al. 2017 ). The lobed leaf trait in melon is regulated by a single recessive gene, PLL , and only one putative gene ( MELO3C010784 ) has been predicted in the genomic region (Gao et al. 2014 ). However, genetic studies identifying genes that regulate lobed leaves in wax gourds are lacking. Therefore, to fill in this knowledge gap, the present study aimed to elucidate the molecular basis of deeply lobed leaves in wax gourd ( Benincasa hispida ) using quantitative trait loci (QTL) mapping, in addition to functional, phylogenetic, and expression analyses. Materials and methods 2.1 Plant materials, phenotyping, and data processing The parental (P) wax gourds, GX-7 and my-1, were characterized by shallow and deeply lobed leaves, respectively. Planting was completed in May 2023 in a Yong’an wax gourd experimental field (Nanning, Guangxi, China), involving 107 genealogies and including the parents. A field-based randomized block design with three replicates was used. Conventional open-field cultivation and management practices were followed. All materials used in this study were provided by the Nanning Kenong Seedling Corporation (Guangxi, China). The plants were periodically observed; 10 complete leaves were randomly selected from each of the biparental plants, and the growth and development of nodes 15–17 were monitored on days 0, 3, 6, 9, 12, 15, 18, 21, 26, and 31. A leaf sample was collected using a Vernier caliper, and the split depth and lateral vein length, as well as the ratio of the split depth to the lateral vein length (L-S/L), were calculated (Fig. 1 ). Phenotypic data were analyzed using Microsoft Excel 2019 for descriptive statistics; t -tests and analysis of variance were performed using SPSS V27.0 (SPSS Inc, Chicago, IL, USA). GraphPad Prism 8.0.2 was used to perform a frequency distribution analysis of plant data to determine whether the differences between parents were significant (Supplementary Fig. S1 ). 2.2 Histological analysis Leaf samples were used to compare cytological characteristics of lobed leaves belonging to GX-7 and my-1. These samples were taken from leaves with the same growth stage at the mature stage of both parents (Fig. 2 ) to ensure that the maximum cross-sections were obtained. Sections were stained with toluidine blue. Tissue section images were observed using Case Viewer ( https://www.3dhistech.com/solutions/caseviewer/ ). ImageJ ( https://imagej.nih.gov/ij/ ) was used to observe cell sizes in both parents and to estimate the number of cells. 2.3 DNA extraction After two weeks, fresh young leaves of the parents and F 2 plants were collected in 2-mL centrifuge tubes and stored at − 80°C for DNA extraction. DNA was extracted using the improved hexadecyl trimethyl ammonium bromide (CTAB) method (An et al., 2011), and the concentration and purity of DNA were detected using a K5800 ultra-differential photometer (Kaiao, Beijing, China). The quality of DNA was assessed via 1.2% agarose gel electrophoresis. 2.4 QTL mapping We resequenced the parents and 105 inbred lines. Sequences were compared with reference genomes of wax gourds to detect the mutations, and a high-density genetic linkage map was constructed. High-density genetic maps and detailed phenotypic data were used for QTL mapping. The composite interval mapping (CIM) method and QTL Cartographer (version 1.17j) software were used for QTL mapping. The threshold limit of detection (LOD) value was determined using 1,000 permutation tests. 2.5 Fine mapping of candidate genes Based on the QTL sequencing results, InDel markers were designed and developed in the candidate regions. The polymerase chain reaction (PCR) products were scanned and genotyped via PCR. The PCR-amplified products were separated using 8% polyacrylamide gel electrophoresis and genotyped. The primer sequences for the InDel markers are shown in Table S1 . 2.6 Expression analysis of candidate genes Samples were obtained from the mature leaves of wax gourds. RNA was extracted from seven different parts of each leaf (Fig. 3 ); four were located at the leaf tip (#1–4) and the remaining three were located at the bottom of each sinus (#5–7) at the position of the lobe. Primers were designed using Premier5.0 for reference and candidate genes (Table S1 ). Candidate gene expression levels were detected using qPCR and analyzed using QuantStudio6 Flex software (ThermoFisher, Waltham, MA, USA), with actin as the internal control. Relative expression levels were measured using the 2 −∆∆CT method, with RG as the internal reference gene. GraphPad Prism 8.0.2 was used to analyze significant differences between relative expression levels. 2.7 Cloning and sequence analysis of candidate genes Cloning primers were designed using Premier5.0 software according to the gene coding sequence (Table S1 ). Total RNA was extracted from the tender leaves of two parents, GX-7 and my-1, using an Eastern® Super Total RNA Extraction Kit (Promega, Beijing, China) according to the manufacturer's instructions. The sequencing was performed by Shenggong Biotechnology Co., Ltd. (Shanghai, China) and alignments were generated using DNAMANV.6 based on DNA and amino acid sequences. 2.8 Homologous gene identification and phylogenetic analysis of Bch04G012650 The protein sequence encoded by BhDDL4.1 was analyzed using NCBI BLAST (NCBI, Bethesda, MD, USA). Additionally, the protein sequences of homologs in Cucurbitaceae , yam, and turnip were downloaded from NCBI in the FASTA format (Table S2 ). We constructed a phylogenetic tree using the neighbor-joining method (NJM) and performed 1000 bootstrap analyses (MEGA7.0 software, https://www.megasoftware.net/ ). 2.9 Molecular marker validation Molecular marker-assisted selection experiments were conducted using the parents, F 1 generation plants, and 60 extreme wax gourd germplasm resources (50 with shallowly lobed leaves and 10 with deeply lobed leaves). Insertion or deletion markers (InDels) were developed based on coding sequence (CDS) differences between the candidate genes of parents. Premier5.0 (Premier, Canada) was used to design primers (Table S1 ). Results 3.1 Phenotypic analysis The split depth to lateral vein length (L-S/L) ratios differed significantly between GX-7 and my-1 (Fig. 4 a). Therefore, GX-7 and my-1 could be considered suitable parents for constructing recombinant inbred line populations for QTL mapping and mining in relation to lobed leaves. GX-7 exhibited a lobed leaf, whereas my-1 exhibited a deeply lobed leaf. During leaf growth and development, the growth trends of GX-7 and my-1 were essentially the same (Fig. 4 b); however, during the early stage, growth was more rapid in the male parent, GX-7, than in the female parent, my-1. After 3 d, the ratio of development of lobed leaves in GX-7 was less than that in my-1. A slight difference was observed in growth rates when the L-S/L ratios of GX-7 and my-1 were compared during the early stages of leaf growth (0–9 d). The development of lobed leaves of GX-7 and my-1 tended to stabilize in GX-7 and my-1 at 15–31 d. As shown in Fig. 4 , lobed leaves formed rapidly during the early stages of leaf growth and development in wax gourds. The development of deep and shallow lobed leaves was stable during the middle and later stages of leaf growth and development, with the degree of lobed depth tending to stabilize. 3.2 Histological analysis Paraffin sections of the GX-7 and my-1 transverse leaf planes with evident leaf lobes (nos. 6 and 7) were studied at 30 d. The cross-section of the leaf included the cuticle, upper epidermis, palisade tissue, parenchyma cells, stomata, gas channels, veins, and lower epidermis. The size and number of parenchyma cells in transverse sections 6 and 7 differed significantly between GX-7 and my-1. Furthermore, my-1 had a larger number of round parenchymal cells (Fig. 5 a, b), while GX-7 had small, round parenchyma with fewer cells (Fig. 5 c, d). The parents showed significant differences in the number of cells in leaf tissue Nos. 6 and 7 (Fig. 5 e). 3.3 QTL mapping Based on the constructed high-density genetic map (total length 1357.145 cM, average distance between adjacent markers 0.002 cM, and maximum interval between adjacent markers 19.647 cM) and phenotypic values, QTL mapping of the leaf lobes of phenotypic traits, i.e., depth, was performed via the CIM method. On Chr04, one peak exceeded the correlation threshold (Fig. 6 a), revealing a single region (38,779,807–41,241,642) with a total length of 2.46 Mb. The maximum LOD was 4.653, and the phenotypic variation explained (PVE) was 13.60% (> 10%). Therefore, the QTL for lobed leaf was stable. 3.4 Fine mapping of candidate genes QTL mapping showed that the candidate genes regulating lobed leaves were located within the Chr04 interval (Fig. 6 a). Using the reference genome GX-19 and parental resequencing data, the candidate genes were sequenced and annotated. Seven InDel markers were developed for the candidate intervals (Table S1 ), and fine mapping between two of these markers, InDel980 and InDel853, narrowed the candidate region to 1.129 Mb (Fig. 6 b). The results showed that 48 candidate genes were localized to the region; functional annotations were obtained for 44 genes, and 11 genes had nonsynonymous mutations (Table 1 ). Real-time fluorescence quantitative analysis indicated that a significant difference in expression levels in the lobed leaf sites between the parents was only observed for BhDDL4.1, which was annotated as a calcium- and calmodulin-dependent serine and threonine protein kinase that regulates kinase activity and participates in many key intracellular signaling pathways. It also promotes cell metabolism, cell polarity, and DNA damage. The preliminary results of functional annotation and sequence alignment indicated that BhDDL4.1 may play a key role in the regulation of lobed leaves in wax gourds. Table 1 List of candidate genes within the precise localization region of Chr04 Genes Location Nonsynonymous Mutations in Coding Sequences annotation Bch04G012560 Chr04:40,135,450 − 40,142,061 (-) Yes putative CCA tRNA nucleotidyltransferase 2 Bch04G012570 Chr04:40,146,296 − 40,149,834 (-) No alpha-(1,4)-fucosyltransferase Bch04G012580 Chr04:40,153,408 − 40,160,839 (-) Yes uncharacterized protein LOC103496554 isoform X2 Bch04G012590 Chr04:40,213,209 − 40,218,827 (+) Yes receptor protein-tyrosine kinase CEPR2 Bch04G012600 Chr04:40,220,051 − 40,225,413 (-) Yes adenylate kinase, chloroplastic Bch04G012610 Chr04:40,227,371 − 40,228,877 (+) Yes protein phosphatase 1 regulatory subunit INH3-like Bch04G012620 Chr04:40,229,048 − 40,230,468 (-) Yes protein EPIDERMAL PATTERNING FACTOR 1 Bch04G012630 Chr04:40,234,655 − 40,234,937 (-) Yes ATP synthase CF1 beta subunit Bch04G012640 Chr04:40,277,451 − 40,281,723 (+) Yes uncharacterized protein LOC101215032 Bch04G012650 Chr04:40,284,809 − 40,288,848 (-) Yes calcium and calcium/calmodulin-dependent serine/threonine-protein kinase-like Bch04G012660 Chr04:40,293,414 − 40,295,250 (-) Yes ETHYLENE INSENSITIVE 3-like 1 protein isoform X1 Bch04G012680 Chr04:40,319,049 − 40,320,020 (-) Yes uncharacterized protein LOC105435754 Bch04G012690 Chr04:40,320,035–40,324,314 (-) No type I inositol polyphosphate 5-phosphatase 10 isoform X1 Bch04G012700 Chr04:40,333,129 − 40,341,884 (-) No protein SAD1/UNC-84 domain protein 1 Bch04G012710 Chr04:40,347,421 − 40,349,045 (+) No cleavage stimulation factor subunit 50 isoform X1 Bch04G012720 Chr04:40,349,652 − 40,355,953 (-) No DNA polymerase alpha subunit B Bch04G012730 Chr04:40,392,152 − 40,398,099 (-) No ABSCISIC ACID-INSENSITIVE 5-like protein 2 Bch04G012740 Chr04:40,465,769 − 40,469,061 (-) No uncharacterized protein LOC103496536 isoform X1 Bch04G012750 Chr04:40,472,870 − 40,476,877 (-) No transcription factor GAMYB Bch04G012770 Chr04:40,526,402 − 40,532,998 (+) No uncharacterized protein LOC103496534 isoform X2 Bch04G012780 Chr04:40.561,581 − 40,561,897 (+) No hypothetical protein Csa_005338 Bch04G012790 Chr04:40,562,149 − 40,566,622 (+) No phospholipid-transporting ATPase 1 Bch04G012800 Chr04:40,569,769 − 40,574,508 (+) No probable mitochondrial import inner membrane translocase subunit TIM21 isoform X1 Bch04G012810 Chr04:40,576,718 − 40,581,899 (-) No probable inactive leucine-rich repeat receptor-like protein kinase At3g03770 isoform X2 Bch04G012830 Chr04:40,584,452 − 40,584,770 (-) No hypothetical protein FNV43_RR08242 Bch04G012840 Chr04:40,588,295 − 40,600,524 (-) No chlorophyll(ide) b reductase NOL, chloroplastic isoform X1 Bch04G012850 Chr04:40,639,011–40,643,724 (-) No MAP7 domain-containing protein 1-like Bch04G012860 Chr04:40,647,775 − 40,648,785 (-) No hypothetical protein Csa_005322 Bch04G012870 Chr04:40,784,746 − 40,793,265 (+) No nuclear pore complex protein NUP1-like Bch04G012880 Chr04:40,820,754 − 40,822,939 (+) No uncharacterized protein At2g39795, mitochondrial Bch04G012890 Chr04:40,825,902 − 40,826,979 (-) No uncharacterized protein At3g28850 Bch04G012910 Chr04:40,846,472 − 40,847,543 (+) No uncharacterized protein LOC103496524 Bch04G012920 Chr04:40,983,374 − 40,986,936 (-) No protein SMAX1-LIKE 3-like Bch04G012930 Chr04:40,990,180 − 40,990,975 (-) No hypothetical protein Csa_005443 Bch04G012940 Chr04:40,996,277 − 41,001,266 (-) No uncharacterized protein E5676_scaffold304G001060 Bch04G012950 Chr04:41,012,533 − 41,013,029 (-) No hypothetical protein E6C27_scaffold24G001560 Bch04G012960 Chr04:41,049,074 − 41,050,610 (-) No 40S ribosomal protein S17-3 Bch04G012980 Chr04:41,067,711 − 41,069,639 (-) No pentatricopeptide repeat-containing protein At5g04780, mitochondrial Bch04G012990 Chr04:41,106,333 − 41,110,776 (-) No cationic amino acid transporter 6, chloroplastic Bch04G013000 Chr04:41,157,122 − 41,158,799 (+) No transcription factor DIVARICATA Bch04G013010 Chr04:41,182,633 − 41,185,045 (-) No uncharacterized protein LOC103496516 Bch04G013020 Chr04:41,224,854 − 41,228,790 (+) No multiple myeloma tumor-associated protein 2 homolog Bch04G013030 Chr04:41,233,167 − 41,241,111 (+) No uncharacterized protein At2g38710-like Bch04G013040 Chr04:41,253,437 − 41,256,706 (+) No vesicle-associated membrane protein 727 3.5 qPCR analysis of candidate genes To further identify candidate genes, qPCR was used to detect the spatiotemporal expression of GX-7 and my-1 at distinct positions during the mature stage, characterized by deeply lobed leaves. Among 48 candidate genes, only BhDDL4.1 showed a significant expression difference among distinct positions on lobed leaves, whereas the other genes showed no significant differences. The expression levels across seven parts of the leaves are shown in Fig. 7 b. Significant differences were observed between the parent lines in the position seven, where the expression level of BhDDL4.1 in GX-7 was significantly higher than that in my-1 at each leaf tip (#1–4) in leaf tissues 5, 6, and 7. The expression of GX-7 was higher than that of my-1 at all seven positions. A highly significant difference was observed between the expression levels of BhDDL4.1 in the most evident part (#7) of the deeply lobed leaves in parents. These data indicated that the differential expression of BhDDL4.1 may play a key role in the formation of lobed leaves in wax gourds. 3.6 Molecular marker validation An InDel marker (InDel623) was developed via insertion into BhDDL4.1 . Genotypic and phenotypic concordance was examined using 60 pure wax gourd samples with extreme traits, as well as their parents and plants in the F 1 generation. Among 60 samples, ten had deeply lobed leaves and 50 had lobed leaves. The results showed that 38 genotypic bands in lobed samples were consistent with GX-7 phenotypic bands and seven genotypic bands for deeply lobed samples were consistent with my-1 phenotypic bands (Fig. 8 ). A total of 12 genotypic bands inconsistent with the GX-7 phenotypic band and three genotypic bands inconsistent with the my-1 phenotypic band were observed. F1 was the parent, and the genotype–phenotype coincidence rate was 74.03%. 3.7 Phylogenetic analysis To analyze the relationship between BhDDL4.1 and homologous proteins, we obtained protein sequences via NCBI BLAST and generated a phylogenetic tree using the NJM and 1,000 bootstrap replicates based on the sequences from wax gourd and other cucurbits, as well as from yam and turnip. The phylogenetic tree showed that BhDDL4.1 was closely related to XP_031743737.1 in melon and KAA0031691 in cucumber (Fig. 9 ) and more distantly related to homologs in pumpkin and silver-seeded cucumber. Discussion 4.1 Growth and development of lobed leaves in wax gourd The results of this study indicated that the early period of leaf growth and development is crucial for the rapid formation of lobed leaves. Leaf shape morphogenesis is a multifaceted process and has a polygenic basis (Sluis and Hake 2015 ). In the present study, histological observations of transverse sections of mature parental leaves revealed differences in cell size and number between GX-7 and my-1. A significant difference was observed in the number of thin-walled cells at the visible sites of lobed leaves (nos. 6 and 7) compared with that in parental leaves (Fig. 5 e). With respect to cytology, leaf growth and development are mainly regulated by cell proliferation and expansion (Zhang et al. 2021b ), and these processes are regulated by many functionally interrelated genes. In Arabidopsis , the following six gene modules control leaf growth and development and participate in cell proliferation: DA1-DA1 enhancer (EOD1); growth regulatory factor (GRF)-GRF interaction factor (GIF); SWITCH/sucrose non-fermentation (SWI/SNF); gibberellin (GA)-DELLA; KLU; and PEAPOD (Vercruysse et al. 2019 ). In poplar, GRFs finely control the leaf size by regulating cell proliferation and expansion. Further studies are needed to determine whether BhDDL4.1 , which regulates the lobed leaves in wax gourd, regulates the cell size and proliferation. 4.2 Molecular mechanism underlying the regulation of lobed leaves in wax gourd by BhDDL4.1 Bch04G012650 (BhDDL4.1 ) may play the leading role in regulation of lobed leaves in wax gourd. The results of qPCR analysis of seven different parts of the leaf were consistent with those of previously reported qPCR results of candidate genes in lobed leaves in zucchini (Bo et al. 2022a ). In this study, the functional annotation of candidate genes showed that BhDDL4.1 may promote the interaction between calcium and calcium or calmodulin-dependent threonine protein kinases, which regulate kinase activity and participate in several key intracellular signal transduction pathways. In plant cells, Ca 2+ engages in many signal transduction processes, including those involving environmental stimuli. As a second messenger in eukaryotic cells (Li et al., 2024), Ca 2+ regulates cell division and apoptosis and plays an important role in regulating various cellular processes (Islam 2020 ); (Rosendo-Pineda et al. 2020 ). Calmodulin, an important mediator of calcium in the cell cycle, regulates numerous kinases, such as cyclin dependent kinase (CDK1) (Berridge et al. 2000 ), as well as the phosphatases involved in these processes. It also interacts with many other cellular proteins and plays a signaling role in various cells (Moser et al. 1995 ); (Park et al. 2019 ). During mitosis, CDK1 regulates and reduces the activity of Orai1 by phosphorylating STIM1. During mitosis, proteases, protein kinases, and protein phosphatases are highly regulated during transitions between stages, thereby ensuring precise cell division and smooth cell cycle progression (Tyson et al., 2008; (Nurse 2000 ). The intracellular Ca 2+ concentration is strictly and precisely regulated via a complex calcium homeostasis system consisting of various calcium channels, calcium pumps, and calcium antiporters (Cyert and Philpott 2013 ). This finding indicates the existence of a close interaction between calcium ions and threonine protein kinase. Calcium ions regulate the activity of threonine protein kinase, thereby affecting its downstream signaling process; alternatively, threonine acid protein kinase regulates the activity of calcium ion channels via phosphorylation, further affecting concentration of intracellular Ca 2+ . Such interactions enable plants to finely regulate physiological functions coping with complex external environments. Declarations All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Funding Guangxi Science and Technology Program, AB21220029, Operation Spike, AA23062048 Competing Interests The authors declare no conflicts of interest. Authur Contributions ZL, PW and XY conceived and designed the study. WB conducted all experiments. YD, ZC, WY, TL, LN, LS, and ZC participated in some experiments. WB wrote the manuscript. ZL, PW and XY revised the manuscript. All authors reviewed the manuscript. Data Availability The data presented in this study are available in this article and as supplementary materials. References Andres RJ, Bowman DT, Kaur B, Kuraparthy V (2014) Mapping and genomic targeting of the major leaf shape gene (L) in Upland cotton (Gossypium hirsutum L.). 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Supplementary Files supplementarytable.docx Cite Share Download PDF Status: Published Journal Publication published 19 Jul, 2024 Read the published version in Euphytica → Version 1 posted Editorial decision: Revision requested 05 May, 2024 Reviews received at journal 28 Apr, 2024 Reviews received at journal 19 Apr, 2024 Reviewers agreed at journal 08 Apr, 2024 Reviewers agreed at journal 07 Apr, 2024 Reviewers invited by journal 07 Apr, 2024 Submission checks completed at journal 12 Mar, 2024 Editor assigned by journal 12 Mar, 2024 First submitted to journal 12 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4085732","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":278968032,"identity":"a9b6df59-9815-42a2-b1d6-0f454f024573","order_by":0,"name":"Wenhui Bai","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Wenhui","middleName":"","lastName":"Bai","suffix":""},{"id":278968036,"identity":"9bd53c96-f0b6-4ded-bda3-504c165dac6e","order_by":1,"name":"Peng Wang","email":"","orcid":"","institution":"Guangxi 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length\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4085732/v1/ec071c7e079372df38db68ef.png"},{"id":52721533,"identity":"95a9edcb-353f-4c63-b59b-dfb4de437e4e","added_by":"auto","created_at":"2024-03-15 01:45:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":223482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of lobed leaf sampling for GX-7 and my-1\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4085732/v1/1ac4f5d3d56be43cffd7325f.png"},{"id":52721717,"identity":"6aa5b4ce-61bd-4a29-a267-21d03f77a4bf","added_by":"auto","created_at":"2024-03-15 01:53:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":218570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSampling sites used for qPCR\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4085732/v1/eae8b2b05eb51c3ffb3972f6.png"},{"id":52721716,"identity":"e195df8a-c441-42e5-a80d-404a2a73e0ce","added_by":"auto","created_at":"2024-03-15 01:53:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":102734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of phenotypic differences of lobed leaf in wax gourd. (a) difference of the ratio of the split depth to lateral vein length (L-S/L) between GX-7 and my-1 (N: number of leaves measured); **P \u0026lt; 0.01 (t-test); values are expressed as means ± SE; (b) growth curves of split depth/lateral vein length in GX-7 and my-1\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4085732/v1/23c86ead32ce40ec7e1ef448.png"},{"id":52721543,"identity":"0ac12d1b-bef1-454a-8d1c-be2f87ced1d5","added_by":"auto","created_at":"2024-03-15 01:45:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":389846,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell size and cell count analyses of paraffin sections of 30-day leaves of wax gourd in areas of visible foliation: (a) Paraffin section of my-1 leaf at No. 7; (b) Paraffin section of my-1 leaf at No. 6 : (c) Paraffin section of GX-7 leaf at No. 7; (d) Paraffin section of GX-7 leaf at No. 6; scale bar = 0.1 mm; (e) Differential analysis of cell number of GX-7 and my-1 at the two sites (t-test; **P \u0026lt; 0.01 and ***P \u0026lt; 0.001, respectively); values are expressed as means ± SE\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4085732/v1/bc18cb99f28e1bf25e95d2c1.png"},{"id":52721542,"identity":"6ae098eb-556e-4e66-959e-4ffae71671be","added_by":"auto","created_at":"2024-03-15 01:45:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":235262,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantitative trait loci (QTL) mapping: (a) High-density genetic map of Chr04; (b) QTL analysis of Chr04 in lobed leaves (c) Candidate genes within the QTL interval; (d) CDS and protein sequences of BhDDL4.1 in the two parents; * indicates translation termination\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4085732/v1/04fa10474bf2e7583fd39f19.png"},{"id":52721537,"identity":"05bde21e-a96a-40e2-90b2-5fdd0ae2409e","added_by":"auto","created_at":"2024-03-15 01:45:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":120605,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression levels of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBhDDL4.1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in GX-7 and my-1: (a) Seven leaf segments (1–7) from GX-7 and my-1 were sampled for qPCR; (b) Relative expression levels of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBhDDL4.1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in different parts of GX-7 and my-1. (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003et\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-test *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4085732/v1/1b5d07227a098d64d8276bdc.png"},{"id":52721538,"identity":"64a51221-a817-4f17-b39b-b8b17d48f451","added_by":"auto","created_at":"2024-03-15 01:45:57","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":103514,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenotyping of 60 germplasm resources of wax gourd using InDel markers at the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBhDDL4.1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003elocus. P1 and P2, respectively, represent GX-7 and my-1; 1–50 are shallowly lobed leaves and 51–60 are deeply lobed leaves\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4085732/v1/ec7e0fc385a28bed5d06c396.png"},{"id":52721539,"identity":"9feaccda-1a93-4d5e-ab8b-f86256c35438","added_by":"auto","created_at":"2024-03-15 01:45:57","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":46187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree of BhDDL4.1 and homologous proteins. The phylogenetic tree was constructed using MEGA-7 and numbers at the nodes indicate bootstrap support based on 1,000 replicates\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4085732/v1/14ab202b4fe98196ed0556b8.png"},{"id":61595120,"identity":"c5703a47-0f26-4def-9112-00ca7571c0d4","added_by":"auto","created_at":"2024-08-01 17:20:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2705215,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4085732/v1/9faf290d-1d07-482c-8796-0540585100c9.pdf"},{"id":52721541,"identity":"2a459e4d-8a65-44f6-8461-7878d9bd0102","added_by":"auto","created_at":"2024-03-15 01:45:57","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":45982,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarytable.docx","url":"https://assets-eu.researchsquare.com/files/rs-4085732/v1/ef5b0e10da2a578e09fb378c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fine mapping of BhDDL4.1, a major gene controlling the regulation of the deeply lobed leaf trait in wax gourd (Benincasa hispida)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWax gourd (\u003cem\u003eBenincasa hispida\u003c/em\u003e (Thunb.) Cogn.) (2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;24), also known as pillow melon, broad melon, or big gourd, belongs to the family Cucurbitaceae. It is an annual herb originating in southern China and eastern India. It has a wide planting range and a long planting history of cultivation. It is one of the most important vegetables transported from southern to northern China (Hu et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The leaves are responsible for photosynthesis, nutrient distribution, water transport, and gas exchange and are, therefore, considered economically important nutritional organs. Phenotypic differences in leaf shape occur within and between species (Nicotra et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e); (Andres et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e); (Rodriguez et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e); (Jiao et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)). Leaf shape in wax gourd can either be non-cracked, serrated, or deeply lobed (Ni et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Additionally, the leaf shape is affected by genetic factors as well as external conditions, such as temperature and light (Tsukaya \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2005\u003c/span\u003e); (Walter and Schurr \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). It may affect plant growth, development, structure, and function as well as the final quality of fruits (Heng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e); (Karamat et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)). Generally, only the upper leaves of those densely planted plants with larger leaves are fully exposed to sunlight and effectively engage in photosynthesis. The leaves of plants with small leaf areas are not obstructed by adjacent leaves and are, therefore, able to fully absorb sunlight and effectively utilize light energy to improve photosynthetic efficiency. This reduces transpiration in plants (Yates et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010\u003c/span\u003e); (Wilf \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Additionally, leaf shape is associated with plant stress resistance (Lopez-Iglesias et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In practice, crops with deeply lobed leaves are more suitable for high-density cultivation (Tu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Therefore, studies focused on investigation of the regulatory genes linked to wax gourd-lobed leaves may positively impact the efforts aimed at improving germplasm resources, increasing yield, and enhancing economic benefits.\u003c/p\u003e \u003cp\u003eIn recent years, many genes and gene families associated with the development of leaf lobes in plants, such as \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, have been discovered. The class I homeodomain leucine zipper transcription factor LMI1 plays an important role in the formation of serrated lobes in \u003cem\u003eArabidopsis\u003c/em\u003e (Saddic et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006a\u003c/span\u003e). The LMI1HD-ZIP transcription factor contributes to variation in deeply lobed leaves in various crops. Members of the HD-ZipI family participate in many biological processes, including the plant leaf growth and development, leaf polarity establishment, and light signal transduction (Merelo et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e); (Zhou et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e); (Whitewoods et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A comparison of leaf morphology in \u003cem\u003eA. thaliana\u003c/em\u003e and related plants has shown that leaflet development depends on the homology domain protein REDUCED COMPLEXITY (RCO) (Vlad et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Furthermore, the HD-Zip transcription factor GhLMI1-D1b, which is a homolog of LMI1-like genes, was associated with different phenotypic traits in cotton leaf (Andres et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Polymorphic LMI1-like genes regulate the growth and development of watermelon and rapeseed (Wang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Lobed leaves are modulated by the incompletely dominant gene \u003cem\u003eBnLLA10\u003c/em\u003e in rapeseed and the homologous gene \u003cem\u003eLOBED-LEAF1\u003c/em\u003e (\u003cem\u003eBnLL1)\u003c/em\u003e in cabbage (Ni et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); (Hu et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). At the same time, both of these genes can regulate the inheritance of deep lobed leaf in kale (Ni et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e); (Hu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Feather leaf traits in ornamental cabbage are modulated by the semi-dominant gene \u003cem\u003eBoFL\u003c/em\u003e, which encodes an alpha-1,2-glucosyltransferase in \u003cem\u003eArabidopsis\u003c/em\u003e (Feng et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). BrLMI1 is an incompletely dominant positive regulator of lobed leaf formation in non-heading cabbage (Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As a meristem identity regulator, LMI1 plays an important role in regulating the growth and development of meristems, leaf, or bract (Saddic et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2006b\u003c/span\u003e). In cotton, genes that regulate lobed leaves belong to \u003cem\u003eLMI1/RCO\u003c/em\u003e and \u003cem\u003eKNOXI\u003c/em\u003e (Chang et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). RCO regulates the activity of cytokinins, thereby affecting leaf shape parameters (Hajheidari et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), while \u003cem\u003eKnoX1\u003c/em\u003e and \u003cem\u003eRCO\u003c/em\u003e synergistically regulate the plant leaflet growth (Wang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGenes that regulate lobed leaves have been evaluated in Cucurbitaceae. The deeply lobed leaves of Cucurbitaceae are regulated by a single incompletely dominant gene, \u003cem\u003eCpDll\u003c/em\u003e, which encodes an HD-Zip I transcription factor; in particular, its high expression may be related to the formation of deeply lobed leaves (Bo et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). Moreover, the HD-Zip transcription factor plays a major role in the presence of lobed leaves in watermelon (Xu et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A single dominant gene, \u003cem\u003eClLL1\u003c/em\u003e, regulates the growth and development of leaf lobes in watermelon (Wei et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The lobed leaf trait in melon is regulated by a single recessive gene, \u003cem\u003ePLL\u003c/em\u003e, and only one putative gene (\u003cem\u003eMELO3C010784\u003c/em\u003e) has been predicted in the genomic region (Gao et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, genetic studies identifying genes that regulate lobed leaves in wax gourds are lacking. Therefore, to fill in this knowledge gap, the present study aimed to elucidate the molecular basis of deeply lobed leaves in wax gourd (\u003cem\u003eBenincasa hispida\u003c/em\u003e) using quantitative trait loci (QTL) mapping, in addition to functional, phylogenetic, and expression analyses.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant materials, phenotyping, and data processing\u003c/h2\u003e \u003cp\u003eThe parental (P) wax gourds, GX-7 and my-1, were characterized by shallow and deeply lobed leaves, respectively. Planting was completed in May 2023 in a Yong\u0026rsquo;an wax gourd experimental field (Nanning, Guangxi, China), involving 107 genealogies and including the parents. A field-based randomized block design with three replicates was used. Conventional open-field cultivation and management practices were followed. All materials used in this study were provided by the Nanning Kenong Seedling Corporation (Guangxi, China).\u003c/p\u003e \u003cp\u003eThe plants were periodically observed; 10 complete leaves were randomly selected from each of the biparental plants, and the growth and development of nodes 15\u0026ndash;17 were monitored on days 0, 3, 6, 9, 12, 15, 18, 21, 26, and 31. A leaf sample was collected using a Vernier caliper, and the split depth and lateral vein length, as well as the ratio of the split depth to the lateral vein length (L-S/L), were calculated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Phenotypic data were analyzed using Microsoft Excel 2019 for descriptive statistics; \u003cem\u003et\u003c/em\u003e-tests and analysis of variance were performed using SPSS V27.0 (SPSS Inc, Chicago, IL, USA). GraphPad Prism 8.0.2 was used to perform a frequency distribution analysis of plant data to determine whether the differences between parents were significant (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Histological analysis\u003c/h2\u003e \u003cp\u003eLeaf samples were used to compare cytological characteristics of lobed leaves belonging to GX-7 and my-1. These samples were taken from leaves with the same growth stage at the mature stage of both parents (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) to ensure that the maximum cross-sections were obtained. Sections were stained with toluidine blue. Tissue section images were observed using Case Viewer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.3dhistech.com/solutions/caseviewer/\u003c/span\u003e\u003cspan address=\"https://www.3dhistech.com/solutions/caseviewer/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). ImageJ (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to observe cell sizes in both parents and to estimate the number of cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 DNA extraction\u003c/h2\u003e \u003cp\u003eAfter two weeks, fresh young leaves of the parents and F\u003csub\u003e2\u003c/sub\u003e plants were collected in 2-mL centrifuge tubes and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for DNA extraction. DNA was extracted using the improved hexadecyl trimethyl ammonium bromide (CTAB) method (An et al., 2011), and the concentration and purity of DNA were detected using a K5800 ultra-differential photometer (Kaiao, Beijing, China). The quality of DNA was assessed via 1.2% agarose gel electrophoresis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 QTL mapping\u003c/h2\u003e \u003cp\u003eWe resequenced the parents and 105 inbred lines. Sequences were compared with reference genomes of wax gourds to detect the mutations, and a high-density genetic linkage map was constructed. High-density genetic maps and detailed phenotypic data were used for QTL mapping. The composite interval mapping (CIM) method and QTL Cartographer (version 1.17j) software were used for QTL mapping. The threshold limit of detection (LOD) value was determined using 1,000 permutation tests.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Fine mapping of candidate genes\u003c/h2\u003e \u003cp\u003eBased on the QTL sequencing results, InDel markers were designed and developed in the candidate regions. The polymerase chain reaction (PCR) products were scanned and genotyped via PCR. The PCR-amplified products were separated using 8% polyacrylamide gel electrophoresis and genotyped. The primer sequences for the InDel markers are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Expression analysis of candidate genes\u003c/h2\u003e \u003cp\u003eSamples were obtained from the mature leaves of wax gourds. RNA was extracted from seven different parts of each leaf (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e); four were located at the leaf tip (#1\u0026ndash;4) and the remaining three were located at the bottom of each sinus (#5\u0026ndash;7) at the position of the lobe. Primers were designed using Premier5.0 for reference and candidate genes (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Candidate gene expression levels were detected using qPCR and analyzed using QuantStudio6 Flex software (ThermoFisher, Waltham, MA, USA), with actin as the internal control. Relative expression levels were measured using the 2\u003csup\u003e\u0026minus;∆∆CT\u003c/sup\u003e method, with \u003cem\u003eRG\u003c/em\u003e as the internal reference gene. GraphPad Prism 8.0.2 was used to analyze significant differences between relative expression levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cloning and sequence analysis of candidate genes\u003c/h2\u003e \u003cp\u003eCloning primers were designed using Premier5.0 software according to the gene coding sequence (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Total RNA was extracted from the tender leaves of two parents, GX-7 and my-1, using an Eastern\u0026reg; Super Total RNA Extraction Kit (Promega, Beijing, China) according to the manufacturer's instructions. The sequencing was performed by Shenggong Biotechnology Co., Ltd. (Shanghai, China) and alignments were generated using DNAMANV.6 based on DNA and amino acid sequences.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Homologous gene identification and phylogenetic analysis of Bch04G012650\u003c/h2\u003e \u003cp\u003eThe protein sequence encoded by \u003cem\u003eBhDDL4.1\u003c/em\u003e was analyzed using NCBI BLAST (NCBI, Bethesda, MD, USA). Additionally, the protein sequences of homologs in \u003cem\u003eCucurbitaceae\u003c/em\u003e, yam, and turnip were downloaded from NCBI in the FASTA format (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). We constructed a phylogenetic tree using the neighbor-joining method (NJM) and performed 1000 bootstrap analyses (MEGA7.0 software, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.megasoftware.net/\u003c/span\u003e\u003cspan address=\"https://www.megasoftware.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Molecular marker validation\u003c/h2\u003e \u003cp\u003eMolecular marker-assisted selection experiments were conducted using the parents, F\u003csub\u003e1\u003c/sub\u003e generation plants, and 60 extreme wax gourd germplasm resources (50 with shallowly lobed leaves and 10 with deeply lobed leaves). Insertion or deletion markers (InDels) were developed based on coding sequence (CDS) differences between the candidate genes of parents. Premier5.0 (Premier, Canada) was used to design primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Phenotypic analysis\u003c/h2\u003e \u003cp\u003eThe split depth to lateral vein length (L-S/L) ratios differed significantly between GX-7 and my-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Therefore, GX-7 and my-1 could be considered suitable parents for constructing recombinant inbred line populations for QTL mapping and mining in relation to lobed leaves. GX-7 exhibited a lobed leaf, whereas my-1 exhibited a deeply lobed leaf. During leaf growth and development, the growth trends of GX-7 and my-1 were essentially the same (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb); however, during the early stage, growth was more rapid in the male parent, GX-7, than in the female parent, my-1. After 3 d, the ratio of development of lobed leaves in GX-7 was less than that in my-1. A slight difference was observed in growth rates when the L-S/L ratios of GX-7 and my-1 were compared during the early stages of leaf growth (0\u0026ndash;9 d). The development of lobed leaves of GX-7 and my-1 tended to stabilize in GX-7 and my-1 at 15\u0026ndash;31 d. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, lobed leaves formed rapidly during the early stages of leaf growth and development in wax gourds. The development of deep and shallow lobed leaves was stable during the middle and later stages of leaf growth and development, with the degree of lobed depth tending to stabilize.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Histological analysis\u003c/h2\u003e \u003cp\u003eParaffin sections of the GX-7 and my-1 transverse leaf planes with evident leaf lobes (nos. 6 and 7) were studied at 30 d. The cross-section of the leaf included the cuticle, upper epidermis, palisade tissue, parenchyma cells, stomata, gas channels, veins, and lower epidermis. The size and number of parenchyma cells in transverse sections 6 and 7 differed significantly between GX-7 and my-1. Furthermore, my-1 had a larger number of round parenchymal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b), while GX-7 had small, round parenchyma with fewer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d). The parents showed significant differences in the number of cells in leaf tissue Nos. 6 and 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 QTL mapping\u003c/h2\u003e \u003cp\u003eBased on the constructed high-density genetic map (total length 1357.145 cM, average distance between adjacent markers 0.002 cM, and maximum interval between adjacent markers 19.647 cM) and phenotypic values, QTL mapping of the leaf lobes of phenotypic traits, i.e., depth, was performed via the CIM method. On Chr04, one peak exceeded the correlation threshold (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), revealing a single region (38,779,807\u0026ndash;41,241,642) with a total length of 2.46 Mb. The maximum LOD was 4.653, and the phenotypic variation explained (PVE) was 13.60% (\u0026gt;\u0026thinsp;10%). Therefore, the QTL for lobed leaf was stable.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Fine mapping of candidate genes\u003c/h2\u003e \u003cp\u003eQTL mapping showed that the candidate genes regulating lobed leaves were located within the Chr04 interval (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Using the reference genome GX-19 and parental resequencing data, the candidate genes were sequenced and annotated. Seven InDel markers were developed for the candidate intervals (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and fine mapping between two of these markers, InDel980 and InDel853, narrowed the candidate region to 1.129 Mb (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The results showed that 48 candidate genes were localized to the region; functional annotations were obtained for 44 genes, and 11 genes had nonsynonymous mutations (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Real-time fluorescence quantitative analysis indicated that a significant difference in expression levels in the lobed leaf sites between the parents was only observed for BhDDL4.1, which was annotated as a calcium- and calmodulin-dependent serine and threonine protein kinase that regulates kinase activity and participates in many key intracellular signaling pathways. It also promotes cell metabolism, cell polarity, and DNA damage. The preliminary results of functional annotation and sequence alignment indicated that BhDDL4.1 may play a key role in the regulation of lobed leaves in wax gourds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of candidate genes within the precise localization region of Chr04\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNonsynonymous Mutations in Coding Sequences\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eannotation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,135,450\u0026thinsp;\u0026minus;\u0026thinsp;40,142,061 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eputative CCA tRNA nucleotidyltransferase 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,146,296\u0026thinsp;\u0026minus;\u0026thinsp;40,149,834 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ealpha-(1,4)-fucosyltransferase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,153,408\u0026thinsp;\u0026minus;\u0026thinsp;40,160,839 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003euncharacterized protein LOC103496554 isoform X2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012590\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,213,209\u0026thinsp;\u0026minus;\u0026thinsp;40,218,827 (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ereceptor protein-tyrosine kinase CEPR2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,220,051\u0026thinsp;\u0026minus;\u0026thinsp;40,225,413 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eadenylate kinase, chloroplastic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012610\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,227,371\u0026thinsp;\u0026minus;\u0026thinsp;40,228,877 (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eprotein phosphatase 1 regulatory subunit INH3-like\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012620\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,229,048\u0026thinsp;\u0026minus;\u0026thinsp;40,230,468 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eprotein EPIDERMAL PATTERNING FACTOR 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,234,655\u0026thinsp;\u0026minus;\u0026thinsp;40,234,937 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eATP synthase CF1 beta subunit\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,277,451\u0026thinsp;\u0026minus;\u0026thinsp;40,281,723 (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003euncharacterized protein LOC101215032\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012650\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,284,809\u0026thinsp;\u0026minus;\u0026thinsp;40,288,848 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecalcium and calcium/calmodulin-dependent serine/threonine-protein kinase-like\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,293,414\u0026thinsp;\u0026minus;\u0026thinsp;40,295,250 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eETHYLENE INSENSITIVE 3-like 1 protein isoform X1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012680\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,319,049\u0026thinsp;\u0026minus;\u0026thinsp;40,320,020 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003euncharacterized protein LOC105435754\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012690\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,320,035\u0026ndash;40,324,314 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003etype I inositol polyphosphate 5-phosphatase 10 isoform X1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,333,129\u0026thinsp;\u0026minus;\u0026thinsp;40,341,884 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eprotein SAD1/UNC-84 domain protein 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012710\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,347,421\u0026thinsp;\u0026minus;\u0026thinsp;40,349,045 (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecleavage stimulation factor subunit 50 isoform X1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012720\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,349,652\u0026thinsp;\u0026minus;\u0026thinsp;40,355,953 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDNA polymerase alpha subunit B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012730\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,392,152\u0026thinsp;\u0026minus;\u0026thinsp;40,398,099 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eABSCISIC ACID-INSENSITIVE 5-like protein 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012740\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,465,769\u0026thinsp;\u0026minus;\u0026thinsp;40,469,061 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003euncharacterized protein LOC103496536 isoform X1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,472,870\u0026thinsp;\u0026minus;\u0026thinsp;40,476,877 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003etranscription factor GAMYB\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012770\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,526,402\u0026thinsp;\u0026minus;\u0026thinsp;40,532,998 (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003euncharacterized protein LOC103496534 isoform X2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012780\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40.561,581\u0026thinsp;\u0026minus;\u0026thinsp;40,561,897 (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehypothetical protein Csa_005338\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012790\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,562,149\u0026thinsp;\u0026minus;\u0026thinsp;40,566,622 (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ephospholipid-transporting ATPase 1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,569,769\u0026thinsp;\u0026minus;\u0026thinsp;40,574,508 (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eprobable mitochondrial import inner membrane translocase subunit TIM21 isoform X1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,576,718\u0026thinsp;\u0026minus;\u0026thinsp;40,581,899 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eprobable inactive leucine-rich repeat receptor-like protein kinase At3g03770 isoform X2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,584,452\u0026thinsp;\u0026minus;\u0026thinsp;40,584,770 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehypothetical protein FNV43_RR08242\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012840\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,588,295\u0026thinsp;\u0026minus;\u0026thinsp;40,600,524 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echlorophyll(ide) b reductase NOL, chloroplastic isoform X1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012850\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,639,011\u0026ndash;40,643,724 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMAP7 domain-containing protein 1-like\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012860\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,647,775\u0026thinsp;\u0026minus;\u0026thinsp;40,648,785 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehypothetical protein Csa_005322\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012870\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,784,746\u0026thinsp;\u0026minus;\u0026thinsp;40,793,265 (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enuclear pore complex protein NUP1-like\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012880\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,820,754\u0026thinsp;\u0026minus;\u0026thinsp;40,822,939 (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003euncharacterized protein At2g39795, mitochondrial\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012890\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,825,902\u0026thinsp;\u0026minus;\u0026thinsp;40,826,979 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003euncharacterized protein At3g28850\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012910\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,846,472\u0026thinsp;\u0026minus;\u0026thinsp;40,847,543 (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003euncharacterized protein LOC103496524\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012920\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,983,374\u0026thinsp;\u0026minus;\u0026thinsp;40,986,936 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eprotein SMAX1-LIKE 3-like\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012930\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,990,180\u0026thinsp;\u0026minus;\u0026thinsp;40,990,975 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehypothetical protein Csa_005443\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012940\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:40,996,277\u0026thinsp;\u0026minus;\u0026thinsp;41,001,266 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003euncharacterized protein E5676_scaffold304G001060\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012950\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:41,012,533\u0026thinsp;\u0026minus;\u0026thinsp;41,013,029 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehypothetical protein E6C27_scaffold24G001560\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:41,049,074\u0026thinsp;\u0026minus;\u0026thinsp;41,050,610 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40S ribosomal protein S17-3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012980\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:41,067,711\u0026thinsp;\u0026minus;\u0026thinsp;41,069,639 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003epentatricopeptide repeat-containing protein At5g04780, mitochondrial\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G012990\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:41,106,333\u0026thinsp;\u0026minus;\u0026thinsp;41,110,776 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecationic amino acid transporter 6, chloroplastic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G013000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:41,157,122\u0026thinsp;\u0026minus;\u0026thinsp;41,158,799 (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003etranscription factor DIVARICATA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G013010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:41,182,633\u0026thinsp;\u0026minus;\u0026thinsp;41,185,045 (-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003euncharacterized protein LOC103496516\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G013020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:41,224,854\u0026thinsp;\u0026minus;\u0026thinsp;41,228,790 (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003emultiple myeloma tumor-associated protein 2 homolog\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G013030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:41,233,167\u0026thinsp;\u0026minus;\u0026thinsp;41,241,111 (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003euncharacterized protein At2g38710-like\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBch04G013040\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChr04:41,253,437\u0026thinsp;\u0026minus;\u0026thinsp;41,256,706 (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003evesicle-associated membrane protein 727\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 qPCR analysis of candidate genes\u003c/h2\u003e \u003cp\u003eTo further identify candidate genes, qPCR was used to detect the spatiotemporal expression of GX-7 and my-1 at distinct positions during the mature stage, characterized by deeply lobed leaves. Among 48 candidate genes, only \u003cem\u003eBhDDL4.1\u003c/em\u003e showed a significant expression difference among distinct positions on lobed leaves, whereas the other genes showed no significant differences. The expression levels across seven parts of the leaves are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb. Significant differences were observed between the parent lines in the position seven, where the expression level of \u003cem\u003eBhDDL4.1\u003c/em\u003e in GX-7 was significantly higher than that in my-1 at each leaf tip (#1\u0026ndash;4) in leaf tissues 5, 6, and 7. The expression of GX-7 was higher than that of my-1 at all seven positions. A highly significant difference was observed between the expression levels of \u003cem\u003eBhDDL4.1\u003c/em\u003e in the most evident part (#7) of the deeply lobed leaves in parents. These data indicated that the differential expression of \u003cem\u003eBhDDL4.1\u003c/em\u003e may play a key role in the formation of lobed leaves in wax gourds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Molecular marker validation\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAn InDel marker (InDel623) was developed via insertion into \u003cem\u003eBhDDL4.1\u003c/em\u003e. Genotypic and phenotypic concordance was examined using 60 pure wax gourd samples with extreme traits, as well as their parents and plants in the F\u003csub\u003e1\u003c/sub\u003e generation. Among 60 samples, ten had deeply lobed leaves and 50 had lobed leaves. The results showed that 38 genotypic bands in lobed samples were consistent with GX-7 phenotypic bands and seven genotypic bands for deeply lobed samples were consistent with my-1 phenotypic bands (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). A total of 12 genotypic bands inconsistent with the GX-7 phenotypic band and three genotypic bands inconsistent with the my-1 phenotypic band were observed. F1 was the parent, and the genotype\u0026ndash;phenotype coincidence rate was 74.03%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Phylogenetic analysis\u003c/h2\u003e \u003cp\u003eTo analyze the relationship between BhDDL4.1 and homologous proteins, we obtained protein sequences via NCBI BLAST and generated a phylogenetic tree using the NJM and 1,000 bootstrap replicates based on the sequences from wax gourd and other cucurbits, as well as from yam and turnip. The phylogenetic tree showed that BhDDL4.1 was closely related to XP_031743737.1 in melon and KAA0031691 in cucumber (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e) and more distantly related to homologs in pumpkin and silver-seeded cucumber.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Growth and development of lobed leaves in wax gourd\u003c/h2\u003e \u003cp\u003eThe results of this study indicated that the early period of leaf growth and development is crucial for the rapid formation of lobed leaves. Leaf shape morphogenesis is a multifaceted process and has a polygenic basis (Sluis and Hake \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, histological observations of transverse sections of mature parental leaves revealed differences in cell size and number between GX-7 and my-1. A significant difference was observed in the number of thin-walled cells at the visible sites of lobed leaves (nos. 6 and 7) compared with that in parental leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). With respect to cytology, leaf growth and development are mainly regulated by cell proliferation and expansion (Zhang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e), and these processes are regulated by many functionally interrelated genes. In \u003cem\u003eArabidopsis\u003c/em\u003e, the following six gene modules control leaf growth and development and participate in cell proliferation: DA1-DA1 enhancer (EOD1); growth regulatory factor (GRF)-GRF interaction factor (GIF); SWITCH/sucrose non-fermentation (SWI/SNF); gibberellin (GA)-DELLA; KLU; and PEAPOD (Vercruysse et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In poplar, GRFs finely control the leaf size by regulating cell proliferation and expansion. Further studies are needed to determine whether \u003cem\u003eBhDDL4.1\u003c/em\u003e, which regulates the lobed leaves in wax gourd, regulates the cell size and proliferation.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4.2 Molecular mechanism underlying the regulation of lobed leaves in wax gourd by\u003c/b\u003e \u003cb\u003eBhDDL4.1\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eBch04G012650 (BhDDL4.1\u003c/em\u003e) may play the leading role in regulation of lobed leaves in wax gourd. The results of qPCR analysis of seven different parts of the leaf were consistent with those of previously reported qPCR results of candidate genes in lobed leaves in zucchini (Bo et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). In this study, the functional annotation of candidate genes showed that BhDDL4.1 may promote the interaction between calcium and calcium or calmodulin-dependent threonine protein kinases, which regulate kinase activity and participate in several key intracellular signal transduction pathways.\u003c/p\u003e \u003cp\u003eIn plant cells, Ca\u003csup\u003e2+\u003c/sup\u003e engages in many signal transduction processes, including those involving environmental stimuli. As a second messenger in eukaryotic cells (Li et al., 2024), Ca\u003csup\u003e2+\u003c/sup\u003e regulates cell division and apoptosis and plays an important role in regulating various cellular processes (Islam \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e); (Rosendo-Pineda et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Calmodulin, an important mediator of calcium in the cell cycle, regulates numerous kinases, such as cyclin dependent kinase (CDK1) (Berridge et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), as well as the phosphatases involved in these processes. It also interacts with many other cellular proteins and plays a signaling role in various cells (Moser et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1995\u003c/span\u003e); (Park et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). During mitosis, CDK1 regulates and reduces the activity of Orai1 by phosphorylating STIM1. During mitosis, proteases, protein kinases, and protein phosphatases are highly regulated during transitions between stages, thereby ensuring precise cell division and smooth cell cycle progression (Tyson et al., 2008; (Nurse \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration is strictly and precisely regulated via a complex calcium homeostasis system consisting of various calcium channels, calcium pumps, and calcium antiporters (Cyert and Philpott \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This finding indicates the existence of a close interaction between calcium ions and threonine protein kinase. Calcium ions regulate the activity of threonine protein kinase, thereby affecting its downstream signaling process; alternatively, threonine acid protein kinase regulates the activity of calcium ion channels via phosphorylation, further affecting concentration of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e. Such interactions enable plants to finely regulate physiological functions coping with complex external environments.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll claims expressed in this article are solely those of the authors\u0026nbsp;and do not necessarily represent those of their affiliated\u0026nbsp;organizations, or those of the publisher, the editors, and the\u0026nbsp;reviewers. Any product that may be evaluated in this article, or\u0026nbsp;claim that may be made by its manufacturer, is not guaranteed or\u0026nbsp;endorsed by the publisher.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGuangxi Science and Technology Program, AB21220029, Operation Spike, AA23062048\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthur Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZL, PW and\u0026nbsp;XY\u0026nbsp;conceived and designed\u0026nbsp;the study. WB conducted all experiments. YD,\u0026nbsp;ZC, WY, TL, LN, LS, and ZC participated in some experiments. WB wrote\u0026nbsp;the manuscript. ZL, PW and\u0026nbsp;XY\u0026nbsp;revised the manuscript. All authors\u0026nbsp;reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in this study are available in this article and as supplementary materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAndres RJ, Bowman DT, Kaur B, Kuraparthy V (2014) Mapping and genomic targeting of the major leaf shape gene (L) in Upland cotton (Gossypium hirsutum L.). 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Thus, determining the molecular mechanisms underlying the development of lobed leaves is important. To this end, in this study, we aimed to resequence 105 recombinant inbred lines, constructed using the parental lines, GX-7 and my-1, to elucidate the molecular mechanisms underlying leaf development in wax gourd (\u003cem\u003eBenincasa hispida\u003c/em\u003e). Genes associated with lobed leaves in wax gourds were first evaluated via quantitative trait loci (QTL) mapping. Next, the F2 population was expanded to 2,000 plants for fine mapping and candidate gene analyses. Thus, the candidate area is reduced to 1.129 Mb, located between the markers InDel980 and InDel853. Functional analyses of candidate genes were performed using gene functional annotation, coding sequence analyses, and expression analyses. Among 48 genes in the candidate region, only \u003cem\u003eBch04G012650 \u003c/em\u003e(termed\u003cem\u003e BhDDL4.1)\u003c/em\u003e showed differences in expression between two parents. Using sequence differences of previously screened candidate genes, an InDel marker (InDel623) was developed in \u003cem\u003eBhDDL4.1\u003c/em\u003e for molecular marker-assisted breeding of wax gourd, and the accuracy rate was 74.03%. Our results indicate that \u003cem\u003eBhDDL4.1\u003c/em\u003e may play a key role in regulation of the lobed leaf trait; thereby, we provided a theoretical basis for further exploration of the molecular mechanisms underlying the lobed leaf trait in wax gourds.\u003c/p\u003e","manuscriptTitle":"Fine mapping of BhDDL4.1, a major gene controlling the regulation of the deeply lobed leaf trait in wax gourd (Benincasa hispida)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-15 01:45:52","doi":"10.21203/rs.3.rs-4085732/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-05T18:56:48+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-28T14:27:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-19T07:43:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b467bdd7-dd70-406c-9c95-0e833b3be764","date":"2024-04-08T23:50:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"482766aa-2dfa-4940-893e-9d093051060b","date":"2024-04-08T01:30:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-07T12:58:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-13T02:44:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-13T02:44:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Euphytica","date":"2024-03-12T13:40:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"euphytica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"euph","sideBox":"Learn more about [Euphytica](https://www.springer.com/journal/10681)","snPcode":"10681","submissionUrl":"https://submission.springernature.com/new-submission/10681/3","title":"Euphytica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"15350f9e-9952-49f9-9f4d-ad644c4a62ce","owner":[],"postedDate":"March 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T16:18:30+00:00","versionOfRecord":{"articleIdentity":"rs-4085732","link":"https://doi.org/10.1007/s10681-024-03381-8","journal":{"identity":"euphytica","isVorOnly":false,"title":"Euphytica"},"publishedOn":"2024-07-19 16:05:23","publishedOnDateReadable":"July 19th, 2024"},"versionCreatedAt":"2024-03-15 01:45:52","video":"","vorDoi":"10.1007/s10681-024-03381-8","vorDoiUrl":"https://doi.org/10.1007/s10681-024-03381-8","workflowStages":[]},"version":"v1","identity":"rs-4085732","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4085732","identity":"rs-4085732","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}