Quantitative trait loci sequencing and genetic mapping reveal two main regulatory genes for stem color in wax gourds

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(2n = 2x = 24)] is a staple vegetable variety in China, especially southern China [ 1 , 2 ]. Stem color is an important agronomic trait of wax gourds; however, its regulatory genes have not been identified. Methods In this study, 105 inbred lines constructed from two parents (GX-71 and MY-1) were sequenced again, and quantitative trait loci sequencing (QTL-seq) was used to mine the genes that regulate stem color in wax gourds. Results Two QTLs related to stem color, qSC5 and qSC12, were identified. QTL localization revealed, for the first time, that the stem color QTL qSC5 and qSC12 are located on Chr05 (11,134,567–16,459,268) and Chr12 (74,618,168–75,712,335), respectively. The explainable phenotypic variation rate and maximum limit of detection(LOD)of qSC5 were 36.9% and 16.9, respectively, while those of qSC12 were 20.9% and 11.2, respectively. Additionally, Bch05G003950 (named BchAPRR2) and Bch12G020400 were identified as candidate genes involved in stem color regulation in wax gourds. Moreover, the chlorophyll content and fluorescence expression levels of BchAPRR2 and Bch12G020400 were significantly higher in green-stemmed wax gourds than those in white-stemmed ones. Therefore, BchAPRR2 and Bch12G020400 were considered the main and secondary regulatory genes for wax gourd stem color, respectively. Finally, InDel markers closely linked to BchAPRR2 were developed to validate the prediction of wax gourd stem color traits in 55 germplasm lines, with an accuracy of 81.8%. Conclusions This study identified the main and secondary genes regulating stem color in wax gourds; these findings lay the foundation for exploring the genetic regulation of wax gourd stem color and future research on wax gourd breeding. wax gourd regulatory gene stem color gene mapping sequencing chlorophyll Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background In 2022, Luo et al. [ 3 ] reported that the stem of wax gourd forms becomes wavy when the BhSAUR60 protein is overexpressed. However, the genes controlling wax gourd stem color have not been identified. In recent years, many genes related to pigment traits of gourds have been revealed. In watermelon and melon; for instance, CmAPRR2 is related to pigment accumulation in fruits [ 4 ]. In balsam pear, the APRR2 gene is located in a 13.87 kb region containing only one predicted gene. The base insertion of the exon leads to a change in the structure of the encoded protein; thus, it could regulate the stigma color of balsam pear as well as the green stigma trait [ 5 ]. HG_GLEAN_10010973 is reportedly associated with the formation of green skin in bottle gourds [ 6 ]. In zucchini, a 14 kb deletion between CP4.1LG15g03360 and CP4.1LG15g0420 , both of which are APRR2 genes, was reported; the deletion of the chromosomal fragment of the repeated locus on APRR2 was reported to affect chlorophyll synthesis in zucchini stems, leading to the production of white-stem varieties of zucchini [ 7 ]. Additionally, EGP19168.1 , the gene regulating the color of eggplant peel, was located in a previous study [ 8 ]. Meanwhile, in wax gourds, the Bch05G003950 gene encodes a bicomponent response regulator-like protein, Arabidopsis pseudo-response regulator 2 (APRR2). Mutations in two bases in this gene lead to the formation of a premature termination codon and the inhibition of chlorophyll development and synthesis, resulting in the formation of white-skin varieties of wax gourds [ 9 ]. Many pigment-related genes have been mapped; however, they are rarely found in stems. Therefore, this study explored the mechanisms underlying the generation of plant pigments. Chlorophyll, carotenoids, and anthocyanins are important plant pigments. Chlorophyll (a and b), found in green tissues of plants helps to capture light energy, is synthesized and located in chloroplasts [ 10 , 11 ]. Changes in chlorophyll and anthocyanin contents can lead to mutations in the genes related to plant leaf color [ 12 , 13 ]. Thus, chlorophyll, as a key factor in photosynthesis, is important for improving crop yield. Therefore, researchers are committed to studying the synthesis and regulatory mechanisms of chlorophyll. For example, in one study, a key single nucleotide polymorphism mutation in CmYGP resulted in yellow–green plant traits in melons. CmYGP encodes a Golden2-like transcription factor that is highly expressed in green tissues. Virus-induced gene silencing further confirmed that CmYGP reduced the number of chloroplasts and chlorophyll content, resulting in the formation of yellow–green melon leaves and fruits [ 14 ]. Studies have shown that chlorophyll and auxins are intertwined in Arabidopsis thaliana ; through ARF7-IAA14 mediation, auxin inhibits the chlorophyll synthesis gene in A. thaliana and subsequently chlorophyll accumulation. These findings provide new insights into the regulatory mechanisms of chlorophyll synthesis and accumulation [ 15 ]. In another study on tomatoes, inhibition of Slym1 promoted chlorophyll decomposition, which changed the leaf color [ 16 ]. This result suggests that downregulation of the SLMYB72 gene of the R2R3MYB subfamily, which regulates the metabolism of chlorophyll, carotenoids, and flavonoids, causes an uneven coloration in tomato fruit [ 17 ]. Several studies have shown that GLKs , TKN2 , TKN4 , and APRR2 are factors related to pigments [ 18 , 19 , 20 ] that regulate the production, differentiation, and function of chloroplasts; play important roles in immune and stress responses; and are important nodes in the regulatory network [ 21 ]. BEL1-LIKE HOMEODOMAIN4 can inhibit the expression of TKN2, which alters the chlorophyll content and chloroplast development in tomatoes [ 22 ]. Although research on pigment genes is diverse, studies on the stem color of cucurbit crops are lacking, and candidate genes regulating stem color need to be identified and analyzed. Therefore, to elucidate the genes regulating stem color in wax gourds, we constructed a high-density genetic map (HDM) of wax gourds using quantitative trait loci sequencing (QTL-seq) and resequencing data of a high-generation inbred line GX-71 (green stem), a high-generation inbred line MY-1 (white stem), and 105 RIL populations (unpublished data). Results Phenotypic and genetic analyses of the stem color The stem colors of the parents, GX-71 and MY-1, used in this study significantly differed. In the field, GX-71, MY-1, and the F1 generation had green, white, and light green stems, respectively (Fig. 1 ); thus, these gourds were selected for constructing populations used in the QTL location mining of stem color-related QTLs and candidate genes. To analyze the heritability of the stem color, an F2 population of 786 individuals was bred from the parents, GX-71 and MY-1. In the F2 population, 449 and 337 strains had green and white stems, respectively. The separation ratio of the F2 population was close to 9:7 (x 2 = 0.244, p = 0.197), indicating that stem color traits may be regulated by the two major effect genes (Table 1 ). Table 1 Distribution of stem color in wax gourd populations Plant type Total no. Green White X 2 (9:7) P GX-71 10 10 - - - MY-1 10 - 10 - - F1 population 10 10 - - - F2 population 786 449 337 0.244 0.19 Determination of pigment content The chlorophyll and carotenoid contents of the parental stems at different developmental stages were measured at 3, 6, 10, 15, 20, 25, 30, and 40 d after transplanting. At 15 d post-transplant, the chlorophyll content of the green-stemmed parent GX-71 gradually increased, whereas that of the white-stemmed parent MY-1 did not change significantly (Fig. 2 A, B). At 40 d post-transplant, the chlorophyll content of GX-71 was more than five times that of MY-1, and the carotenoid content of GX-71 was also significantly higher than that of MY-1 (Fig. 2 C). QTL mapping and candidate gene identification QTL mapping was performed using a high-density genetic linkage map (unpublished data) and phenotypic data. The map contained 128,9176 variation sites and 1,256,985 markers, with a total distance of 1,345.1 cM. Based on the observed phenotypic values of the 105 inbred lines and HDM (unpublished), QTL mapping of stem color-related traits was performed using the CIM method. Two QTLs, qSC5 and qSC12, were identified as the main stable QTLs on 2 chromosomes. Specifically, qSC5 and qSC12 are located on Chr05 (11,134,567–16,459,268) and Chr12 (74,618,168–75,712,335), respectively. The explainable phenotypic variation rate and maximum LOD of qSC5 were 36.9% and 16.9, respectively, while those of qSC12 were 20.9% and 11.2, respectively. Based on the combination of QTL results with unpublished data of the wax gourd reference genome (GX-19) with the GX-71 and MY-1 resequencing data, eight pairs of polymorphic InDel markers were developed for genotyping and linkage analysis of qSC5. Finally, the interval, which contained 14 genes, was narrowed to js13.2 (13,205,354) and js13.8 (13,838,411), with a total length of 633.057 kb (Fig. 3 A). In the constructed F2 population, individuals with different stem color traits without qSC5 exchange were selected for self-pollination, and an F2-1 population of 1,120 individuals was obtained. Four pairs of polymorphic InDel markers were developed to narrow qSC12 to a 569.389 kb region containing only 13 genes (Fig. 3 B). Gene sequence and annotation analyses(Table 2 ) of the genes in this region showed that Bch05G003950 exhibited two-base deletions in the maternal coding sequence, resulting in the formation of a premature termination codon (Fig. 3 A). Additionally, the gene annotation of Bch12G020400 indicated that it encodes phospholipase A (1) DAD1, a protein with chloroplast-like characteristics (Table 3 ). Table 2 Genes within the narrowed qSC5 interval Gene ID Nonsynonymous Mutations in Coding Sequences Physical Location Gene Annotation Bch05G003860 Yes Chr5: 13207272–13212788 (+) protein NRT1/ PTR FAMILY 7.3-like Bch05G003870 Yes Chr5: 13213544–13235133 (-) Pyruvate kinase isozyme G Bch05G003880 Yes Chr5: 13274698–13276057(+) - Bch05G003890 No Chr5: 13276096–13281871(+) Protein ABCI7, chloroplastic Bch05G003900 Yes Chr5: 13341180–13346696(-) Probable ribose-phosphate Pyrophosphokinase 1-like Bch05G003910 Yes Chr5: 13367114–13372139(-) Probable tobamovirus multiplication protein 2B isoform X1 Bch05G003920 Yes Chr5: 13438283–13441573 (-) Probable peroxidase 20 isoform X1 Bch05G003930 No Chr5: 13441626–13447889 (-) Tobamovirus multiplication protein 2A-like Bch05G003940 Yes Chr5: 13484454–13484678 (-) - Bch05G003950 Yes Chr5: 13491669–13499644 (-) Two-component response Regulator-like protein APRR2 Bch05G003960 Yes Chr5: 13676056–13689823 (-) MSC domain-containing protein Bch05G003970 No Chr5: 13805666–13806021 (+) Vacuolar protein sorting-associated protein 55 homolog Bch05G003980 No Chr5: 13810161–13810902 (-) Uncharacterized protein LOC111455443 Bch05G003990 Yes Chr5: 13825779–13838208 (-) Transcription factor MAMYB Table 3 Genes within the narrowed qSC12 interval Gene ID Nonsynonymous Mutations in Coding Sequences Physical Location Gene Annotation Bch12G020370 Yes Chr12: 75155250–75160607 (-) Probable H/ACA ribonucleoprotein complex subunit 1-like Bch12G020380 No Chr12: 75215164–75221452 (-) RGG repeats nuclear RNA binding protein A-like isoform X2 Bch12G020390 No Chr12: 75225625–75297961 (-) Probable glycine–tRNA ligase, chloroplastic/mitochondrial 2 isoform X1- Bch12G020400 No Chr12: 75332840–75335150 (-) Phospholipase A(1) DAD1, chloroplast-like Bch12G020410 No Chr12: 75409841–75418358 (+) Carotenoid cleavage dioxygenase 7, chloroplastic Bch12G020420 No Chr12: 75418509–75419660 (+) Probable major pollen allergen Ole e 6-like Bch12G020430 No Chr12: 75498816–75499589 (+) Probable major pollen allergen Ole e 6-like Bch12G020440 No Chr12: 75510818–75511148 (-) - Bch12G020450 No Chr12: 75522841–75523897 (+) Probable major pollen allergen Ole e 6-like Bch12G020460 No Chr12: 75529773–75530669 (+) Probable major pollen allergen Ole e 6-like Bch12G020470 No Chr12: 75548538–75551483 (-) - Bch12G020480 No Chr12: 75557730–75559794 (-) Pentatricopeptide repeat-containing protein At4g01570 Bch12G020490 No Chr12: 75564027–75565069 (-) Probable uncharacterized protein LOC103498828 qRT-PCR analysis To further analyze the candidate genes, qRT-PCR was performed to examine the expression of genes within the parental region. Stem segments with stable stem color traits were selected from the transplanted 40DAT as materials for differential expression analysis of all genes in the region. Differential expression analysis showed that the expression of only Bch05G003950 and Bch12G020400 differed significantly when compared with those of their parent plants. First, Bch05G003950 exhibited increased relative expression in the parental plant GX-71 and reduced relative expression in the maternal plant MY-1 (Fig. 4A). Second, Bch12G020400 expression was negligible in the maternal plant, but that in the parental plant exhibited significant variation (Fig. 4B). These results indicate that Bch05G003950 and Bch12G020400 may be key genes regulating the stem color in wax gourds. InDel marker-assisted breeding An InDel marker tightly linked to BchAPRR2 was developed to verify the consistency of the stem color genotypes and phenotypes of the experimental wax gourds. In total, 55 wax gourd germplasm resources with extreme stem color traits were selected to verify the InDel marker. Among them, 34 and 21 had green and white stems, respectively. The results showed that 36 and 19 germplasm resources were consistent with green-stem male parent GX-71 bands and white-stem female parent bands, respectively(Fig. 5 ). The coincidence rate of the genotype and phenotype was 81.8%. (Supplementary Table 2). Discussion The stem color of wax gourds is of great significance for their development and fruiting. Differences in chlorophyll content in the stems of wax gourds lead to different stem colors. Numerous studies have been carried out on pigments, but the stem color in Cucurbitaceae has been sparsely investigated. These studies have shown that pigment type and content can determine the color of plant tissues [ 23 ], and that main pigment types can differ by tissue. In some plant tissues, such as the peel of eggplant, chlorophyll and anthocyanins are the primary pigments that determine color [ 24 ]. In Chinese kale, which has purple stems, anthocyanin is the main pigment that governs the stem color; the BoDRF gene, which controls the purple stem color of Chinese kale, is located at a 0.32 cM interval. However, the insertion of a base in this gene leads to a frameshift mutation, resulting in the production of green stems [ 25 ]. Chlorophyll is the main component of green tissues; therefore, a lack of chlorophyll in green tissues leads to plant albinism [ 26 ]. In this study, the main pigment controlling the stem color of wax gourds was determined to be chlorophyll; thus, we observed and measured chlorophyll changes in wax gourd plants for forty days post-transplantation. During this period, the stem chlorophyll contents of parental GX-71 and maternal MY-1 plants significantly differed. Molecular mechanism regulating the stem color of wax gourds Various family factors, including the GLKs, MYB, APRR, and other families, are reportedly involved in the regulation of pigment traits. For example, in strawberries, FaMYB controls the formation of red strawberries by regulating flavonoid biosynthesis during the late stage of fruit development. Wang et al. [ 27 ] inserted eight bases in a specific variant allele of FaMYB10 , which resulted in the formation of a premature termination codon and production of white octoploid strawberries. Additionally, the GLK family transcription factors, which are key nodes in the plant regulatory network, trigger the expression of photosynthesis-related nuclear genes [ 28 ]. For example, the chlorophyll regulatory factor BGP4 is reportedly affects chlorophyll contents by influencing in the conduction of light signals, interaction with the GLK transcription factor, and inhibition of activity of the GLK transcription factor [ 29 ]. In this study, the main regulatory gene for wax gourd stem color was found to be BchAPRR2 , which belongs to the APRR family. In squash, deletion of the chromosomal fragment of the repeated locus on APRR2 leads to the synthesis of chlorophyll in stems, which affects the production of white-stem varieties of squash [ 7 ]. Moreover, APRR1 is related to pigments [ 30 ], and Nong et al. [ 31 ] reported that APRR2 is closely related to the skin color of wax gourds. Furthermore, allele variation in BchAPRR2 across varieties of wax gourds affects the plant chlorophyll content and structure. Moreover, APRR family genes are reportedly involved in the regulation of circadian rhythms in Arabidopsis [ 32 ]. The APRR9 gene of the APRR family encodes regulatory factors related to light-induced photoresponses and participates in the regulation of the circadian rhythm in Arabidopsis [ 33 ]. In this study, annotation of the Bch12G020400 gene revealed that it encodes phospholipase A(1) DAD1, a chloroplast-like protein. In Arabidopsis , the DAD1 protein is chloroplast phospholipase A1, which is a fluorescent green fusion protein mainly located in chloroplasts [ 34 ]. Therefore, high expression of Bch12G020400 may promote the production of the green fluorescent protein DAD1, thereby affecting the stem color of wax gourds. Conclusions In this study, we observed that two-base deletions of the main regulatory gene, APRR2 , of stem color led to a frameshift mutation, which may change the structure and function of the protein. High expression of the secondary regulatory gene Bch12G020400 may promote the production of the green fluorescent protein DAD1 in the chloroplast. BchAPRR2 and Bch12G020400 may affect the synthesis, structure, or function of proteins in chloroplasts via complex molecular regulatory mechanisms, ultimately leading to stem color differences in wax gourds. Two genes, BchAPRR2 and Bch12G020400 , were found to be related to the stem color trait of wax gourd. However, further research on the molecular mechanisms of BchAPRR2 and Bch12G020400 regulating wax gourd stem color may face some difficulties, mainly due to the incomplete establishment of the wax gourd genetic transformation system.Our study provides a theoretical basis for further research on these mechanisms and a reference for stem color regulatory genes of other gourd species, and our findings will help genetically improve wax gourd stem color and specific germplasm resources. Further validation of the molecular mechanisms of BchAPRR2 and Bch12G020400 in regulating wax gourd stem color is needed and can be achieved by combining genetic transformation methods, gene editing, and overexpression techniques. Materials and methods Plant material and phenotypic evaluation In this study, 105 recombinant inbred gourd lines were constructed. Green-stemmed GX-71 (male parent) and white-stemmed MY-1 (female parent) plants were utilized as parents to produce populations for QTL localization. The F1 generation was hybridized, and then the F1 generation individuals were self-pollinated to obtain F2 population, comprising 2,218 plants. Individuals with different stem colors from F2 without qSC5 exchange were selected for self-pollination to obtain the F2-1 line. F2 was used for fine-mapping of genes in qSC5, and F2-1 was used for fine-mapping of genes in qSC12. Simultaneously, 55 germplasm resources were used to verify the InDel markers developed in this study. All materials were procured from Nanning Kenong Seed Industry Co., Ltd., China, and planted at the Nanning Yong'an Wax Gourd Experimental Base (E-108°51', N 22°48') from July 2022 to July 2023 for growth under natural light. The parents of the hybrid combination and F2 generation plants were labeled with serial numbers. The wax gourd plants used for phenotypic determination were collected 40 d after transplantation, which ensured that chlorophyll was in a stable state. The stem color of wax gourds was visually evaluated in the field and categorized as white and green. Specifically, a stem color resembling the white stem of the mother plant was recorded as white, and a color resembling the green stem of the father plant was recorded as green. Extraction and determination of chlorophyll and carotenoid contents Stems were collected at the same plant height at 0, 3, 6, 10, 15, 20, 25, 30, and 40 d after transplanting. The stems were cut into ~ 3 cm-long segments using a knife and ground into a powder with liquid nitrogen. Powder was weighed (1.0 g), placed into a 15 mL centrifuge tube, and shaken in a light-proof environment at 200 rpm for 12 h to extract pigments. The chlorophyll a, chlorophyll b, and carotenoids contents were evaluated at 665, 649, and 470 nm, respectively. The respective equation was derived from Li et al. [ 35 ] (Appendix). DNA extraction A plant genomic DNA extraction kit (Solarbio Science, Beijing, China) was used to extract genomic DNA from the young leaves. The obtained DNA was quantified using an ultra-micro spectrophotometer (K5800, KAIAO, Beijing, China) and its integrity was evaluated using 1.2% agarose gel electrophoresis. QTL mapping and InDel marker analysis QTL mapping was performed using a high-density genetic linkage map (unpublished data) and phenotypic data. The map contained 1,289,176 variation sites, 1,256,985 markers, and had a total distance of 1,345.1 cM. QTL mapping was performed using the composite interval mapping (CIM) method and QTL Cartographer (version 1.17j) software ( https://brcwebportal.cos.ncsu.edu/qtlcart/ ) for preliminary localization. InDel markers were developed for genotyping and linkage analysis using Premier 5.0 software based on the whole-genome resequencing data of the parents and 105 inbred lines. Several developed InDel polymorphic molecular markers were used in combination with the F2 population data for fine-mapping of genes in qSC5. Other developed InDel polymorphic molecular markers were used in combination with F2-1 data for fine-mapping of genes in qSC12. RNA extraction and candidate gene prediction analysis Total RNA was extracted from GX-7 and MY-1 using the EastepSuper Total RNA Extraction Kit (Promega, Beijing, China) according to the manufacturer's instructions. Gene sequence comparisons were performed between the candidate gene sequences, which were obtained via the resequencing of the parents and 105 inbred lines. The corresponding gene annotations were searched in the existing wax gourd reference genome, GX-19. Gene sequence alignments were produced using DNAMAN v.9 software (Lynnon Biosoft, San Ramon, CA, United States). Finally, gene sequence analyses combined with gene annotation analysis were used to screen candidate genes involved in the regulation of wax gourd stem color. qRT-PCR analysis of candidate genes In this study, qRT-PCR was used to quantify the differential expression of candidate genes in the parent plants. First, total RNA was extracted from parental stem segments 40 d after transplantation and reverse-transcribed using a reverse transcriptase RT Master Mix (Takara, Beijing, China). Primer sequences to amplify the reference genes CAC ( Bch05G003650 ), Bch05G003950 , and Bch12G020400 and candidate genes were designed using Premier 5.0 (Supplementary Table 1). The qPCR analysis was performed using a premixed SYBR Green quantitative PCR reagent and an Applied Biosystems 7500 qRT-PCR system (Foster City, CA, USA). Each experiment was repeated three times, and relative expressions were determined using the 2 −∆∆Ct method [ 36 ]. Differences in relative expression were analyzed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). Molecular marker-assisted breeding A pair of InDel markers tightly linked to the predicted main regulatory gene BchAPRR2 was designed using Premier 5.0. The primer sequences are listed in Supplementary Table 1. Combined with developed markers, 55 wax gourd germplasms from the parent plants and F1 with extreme stem color traits, including 34 green stems and 21 white stems, were used for molecular marker-assisted and stem color accuracy verification experiments (Supplementary Table 2). Declarations Funding This research was supported by the Guangxi Science and Technology Base & Talents Fund (Guike AD21220040), National Natural Science Foundation of China (31960593), and the Science and Technology Major Project of Guangxi (Guike AB21220029). Author contributions ZC: Data curation, validation, writing – original draft, formal analysis. PW: methodology, writing – review & editing. WB: Writing – review & editing, data curation, investigation. YD: Data curation, investigation, writing – review & editing. ZKC: Investigation, writing – review & editing, software. LS: Investigation, writing – review & editing. LN: investigation, writing – review & editing. TL: Investigation, writing – review& editing. WY: Investigation, writing – review & editing. XY: Investigation, writing – review & editing, methodology. ZL: Writing – review & editing, methodology, conceptualization. Data Availability The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: CNSA accession number:CNP0004715,https://db.cngb.org/search/?q=CNP0004715. Acknowledgments We are grateful to Guangxi University for providing the experimental instruments for this study. 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The control of red colour by a family of MYB transcription factors in octoploid strawberry ( Fragaria × ananassa ) fruits. Plant Biotechnol J. 2020;18:1169–84. https://doi.org/10.1111/pbi.13282. Zhang T, Zhang R, Zeng XY, Lee S, Ye LH, Tian SL, Zhang YJ, Busch W, Zhou WB, Zhu XG, Wang P. GLK Transcription Factors Accompany ELONGATED HYPOCOTYL5 to Orchestrate Light-induced Seedling Development in Arabidopsis . Plant Physiol. 2024;kiae002. https://doi.org/10.1093/plphys/kiae002. Tachibana R, Abe S, Marugami M, Yamagami A, Akema R, Ohashi T, Nishida K, Nosaki S, Miyakawa T, Tanokura M, Kim JM, Seki M, Inaba T, Matsui M, Ifuku K, Kushiro T, Asami T, Nakano T. BPG4 Regulates Chloroplast Development and Homeostasis by Suppressing GLK Transcription Factors and Involving Light and Brassinosteroid Signaling. Nat Commun. 2024;15:370. https://doi.org/10.1038/s41467-023-44492-5. Pan Y, Bradley G, Pyke K, Ball G, Lu C, Fray R, Marshall A, Jayasuta S, Baxter C, Wijk RV, Boyden L, Cade R, Chapman NH, Fraser PD, Hodgman C, Seymour GB. Network Inference Analysis Identifies an APRR2-Like Gene Linked to Pigment Accumulation in Tomato and Pepper Fruits. Plant Physiol. 2013;161(3):1476–85. https://doi.org/10.1104/pp.112.212654. Nong L, Wang P, Yang W, Liu T, Su L, Cheng Z, Bai W, Deng Y, Chen Z, Liu Z. Analysis of BhAPRR2 allele variation, chlorophyll content, and chloroplast structure of different peel colour varieties of wax gourd ( Benincasa hispida ) and development of molecular markers. Euphytica. 2023;219:107. https://doi.org/10.1007/s10681-023-03233-x. Matsushika A, Makino S, Masaya K, Mizuno T. Circadian Waves of Expression of the APRR1/TOC1 Family of Pseudo-Response Regulators in Arabidopsis Thaliana : Insight into the Plant Circadian Clock. Plant Cell Physiol . 2000;41(9):1002–12. https://doi.org/10.1093/pcp/pcd043. Ito S, Nakamichi N, Matsushika A, Fujimori T, Yamashino T, Mizuno T. Molecular Dissection of the Promoter of the Light-induced and Circadian-controlled APRR9 Gene Encoding a Clock-associated Component of Arabidopsis Thaliana . Biosci Biotech Bioch. 2005;69(2):382–90. https://doi.org/10.1271/bbb.69.382. Ishiguro S, Kawai-Oda A, Ueda J, Nishida I, Okada K. The DEFECTIVE IN ANTHER DEHISCENCE1 Gene Encodes a Novel Phospholipase A1 Catalyzing the Initial Step of Jasmonic Acid Biosynthesis, Which Synchronizes Pollen Maturation, Anther Dehiscence, and Flower Opening in Arabidopsis . Plant Cell. 2001;13(10):2191–209. https://doi.org/10.1105/tpc.010192. Li H. Principles and Techniques of Plant Physiological and Biochemical Experiments. 1st ed. Beijing: Higher Education Press; 2000. Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 − ΔΔ C T Method. Methods. 2001;25:402–8. https://doi.org/10.1006/meth.2001.1262. Additional Declarations No competing interests reported. Supplementary Files Appendix.docx Supplementarytable.xlsx Supplementarytable2.docx supplementaryfile.tiff Cite Share Download PDF Status: Published Journal Publication published 29 Jun, 2024 Read the published version in Plants → Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4134687","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":283962638,"identity":"14405fca-6c8e-415d-b818-7559b10e8323","order_by":0,"name":"Zhihao Chen","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Zhihao","middleName":"","lastName":"Chen","suffix":""},{"id":283962639,"identity":"4731a3c3-38e4-4dee-a93c-a9008642793a","order_by":1,"name":"Peng Wang","email":"","orcid":"","institution":"Guangxi 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Su","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Liwen","middleName":"","lastName":"Su","suffix":""},{"id":283962644,"identity":"0abce25a-b405-4064-a35d-ab3753e71730","order_by":6,"name":"Lifeng Nong","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Lifeng","middleName":"","lastName":"Nong","suffix":""},{"id":283962645,"identity":"21f76fd3-5922-42fb-a006-8d368a230848","order_by":7,"name":"Ting Liu","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Liu","suffix":""},{"id":283962646,"identity":"7ca764d8-e166-4020-8f92-2830b3ab4a82","order_by":8,"name":"Wenrui Yang","email":"","orcid":"","institution":"Guangxi 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06:46:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4134687/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4134687/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.3390/plants13131804","type":"published","date":"2024-06-29T10:34:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53590231,"identity":"6f664d46-542f-4817-bffe-bd9c9a07392b","added_by":"auto","created_at":"2024-03-27 19:58:52","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":72680,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic differences in stem color of the parents, GX-71 and MY-1, and the F1 generation\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4134687/v1/4a02adeb28222fe852a44a24.jpeg"},{"id":53590488,"identity":"65918117-8359-4daf-981f-fbf94a7c855e","added_by":"auto","created_at":"2024-03-27 20:06:53","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":257408,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of parental pigment contents. (A): Chlorophyll a, (B): Chlorophyll b, (C): Chlorophyll, and (D): carotenoid contents. **, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4134687/v1/8f4416c60be3f8a134a36ecf.jpeg"},{"id":53590237,"identity":"9d1da373-0c72-4065-930d-9897345cfb6d","added_by":"auto","created_at":"2024-03-27 19:58:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":424918,"visible":true,"origin":"","legend":"\u003cp\u003eFine-mapping of the main regulatory gene \u003cem\u003eBchAPRR2, \u003c/em\u003eand the secondary gene, \u003cem\u003eBch12G020400,\u003c/em\u003e involved in wax gourd stem color. (A) Fine-mapping of candidate genes in qSC5，which was narrowed to js13.2 and js13.8, with a total length of 633.057 kb, containing 14 genes. Gene sequence analysis of \u003cem\u003eBchAPRR2\u003c/em\u003e revealed two base deletions. (B) Fine-mapping of candidate genes in qSC12; qSC12 was narrowed down to a total length interval of 569.389 kb containing 13 genes.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4134687/v1/a8bbb980f90708949ea8937b.jpg"},{"id":53590238,"identity":"e5fe8075-0dc8-436c-b8b1-8e916f958fe8","added_by":"auto","created_at":"2024-03-27 19:58:53","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":297829,"visible":true,"origin":"","legend":"\u003cp\u003eReal-time quantitative expression of candidate genes. (A) Expression analysis of candidate genes (A) within the reduced qSC5 interval and (B) within the reduced qSC12 interval in stems. **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. “ns,” no significant difference.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4134687/v1/728dd33082fa9a4efcc21e36.jpg"},{"id":53590239,"identity":"00fc2561-d70a-4696-9d40-2395ab6e2e11","added_by":"auto","created_at":"2024-03-27 19:58:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":162197,"visible":true,"origin":"","legend":"\u003cp\u003eInDel marker used to verify the 55 parts of the wax gourd germplasm resources. P1 and P2 represent GX-71 and MY-1, respectively; 1-36 represent green-stemmed wax gourds and 37-55 represent white-stemmed wax gourds.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4134687/v1/384b64d6e8c28a04dde7fc63.png"},{"id":59845536,"identity":"a211ab61-d652-4ae6-973f-e9935651a707","added_by":"auto","created_at":"2024-07-08 10:35:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2057577,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4134687/v1/e560fa32-2018-4bcc-864a-d69d6a2ca5d9.pdf"},{"id":53590487,"identity":"6990aad8-7aeb-4e84-8b72-bbb031596bf3","added_by":"auto","created_at":"2024-03-27 20:06:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15053,"visible":true,"origin":"","legend":"","description":"","filename":"Appendix.docx","url":"https://assets-eu.researchsquare.com/files/rs-4134687/v1/d77a2f0d42036bee5bfeb94d.docx"},{"id":53590234,"identity":"9a4e685f-42b0-48b2-a3f9-3148f7cbce9c","added_by":"auto","created_at":"2024-03-27 19:58:53","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13069,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4134687/v1/1dbb96d12651d5d99dfa5d9a.xlsx"},{"id":53590233,"identity":"87d1f37b-445a-40e1-af0f-b644a05c3d62","added_by":"auto","created_at":"2024-03-27 19:58:53","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":21106,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4134687/v1/ac25737641f8cc7456c62611.docx"},{"id":53590232,"identity":"8a4443d7-10fe-41ff-9861-bd9c3bab69cb","added_by":"auto","created_at":"2024-03-27 19:58:53","extension":"tiff","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":708149,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.tiff","url":"https://assets-eu.researchsquare.com/files/rs-4134687/v1/0ada3a997abfc76b42f2559d.tiff"}],"financialInterests":"No competing interests reported.","formattedTitle":"Quantitative trait loci sequencing and genetic mapping reveal two main regulatory genes for stem color in wax gourds","fulltext":[{"header":"Background","content":"\u003cp\u003eIn 2022, Luo et al. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] reported that the stem of wax gourd forms becomes wavy when the BhSAUR60 protein is overexpressed. However, the genes controlling wax gourd stem color have not been identified. In recent years, many genes related to pigment traits of gourds have been revealed. In watermelon and melon; for instance, \u003cem\u003eCmAPRR2\u003c/em\u003e is related to pigment accumulation in fruits [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In balsam pear, the \u003cem\u003eAPRR2\u003c/em\u003e gene is located in a 13.87 kb region containing only one predicted gene. The base insertion of the exon leads to a change in the structure of the encoded protein; thus, it could regulate the stigma color of balsam pear as well as the green stigma trait [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. HG_GLEAN_10010973 is reportedly associated with the formation of green skin in bottle gourds [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In zucchini, a 14 kb deletion between \u003cem\u003eCP4.1LG15g03360\u003c/em\u003e and \u003cem\u003eCP4.1LG15g0420\u003c/em\u003e, both of which are \u003cem\u003eAPRR2\u003c/em\u003e genes, was reported; the deletion of the chromosomal fragment of the repeated locus on APRR2 was reported to affect chlorophyll synthesis in zucchini stems, leading to the production of white-stem varieties of zucchini [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, \u003cem\u003eEGP19168.1\u003c/em\u003e, the gene regulating the color of eggplant peel, was located in a previous study [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Meanwhile, in wax gourds, the \u003cem\u003eBch05G003950\u003c/em\u003e gene encodes a bicomponent response regulator-like protein, Arabidopsis pseudo-response regulator 2 (APRR2). Mutations in two bases in this gene lead to the formation of a premature termination codon and the inhibition of chlorophyll development and synthesis, resulting in the formation of white-skin varieties of wax gourds [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Many pigment-related genes have been mapped; however, they are rarely found in stems. Therefore, this study explored the mechanisms underlying the generation of plant pigments.\u003c/p\u003e \u003cp\u003eChlorophyll, carotenoids, and anthocyanins are important plant pigments. Chlorophyll (a and b), found in green tissues of plants helps to capture light energy, is synthesized and located in chloroplasts [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Changes in chlorophyll and anthocyanin contents can lead to mutations in the genes related to plant leaf color [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, chlorophyll, as a key factor in photosynthesis, is important for improving crop yield. Therefore, researchers are committed to studying the synthesis and regulatory mechanisms of chlorophyll. For example, in one study, a key single nucleotide polymorphism mutation in \u003cem\u003eCmYGP\u003c/em\u003e resulted in yellow\u0026ndash;green plant traits in melons. \u003cem\u003eCmYGP\u003c/em\u003e encodes a Golden2-like transcription factor that is highly expressed in green tissues. Virus-induced gene silencing further confirmed that CmYGP reduced the number of chloroplasts and chlorophyll content, resulting in the formation of yellow\u0026ndash;green melon leaves and fruits [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Studies have shown that chlorophyll and auxins are intertwined in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e; through ARF7-IAA14 mediation, auxin inhibits the chlorophyll synthesis gene in \u003cem\u003eA. thaliana\u003c/em\u003e and subsequently chlorophyll accumulation. These findings provide new insights into the regulatory mechanisms of chlorophyll synthesis and accumulation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In another study on tomatoes, inhibition of Slym1 promoted chlorophyll decomposition, which changed the leaf color [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This result suggests that downregulation of the \u003cem\u003eSLMYB72\u003c/em\u003e gene of the R2R3MYB subfamily, which regulates the metabolism of chlorophyll, carotenoids, and flavonoids, causes an uneven coloration in tomato fruit [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Several studies have shown that \u003cem\u003eGLKs\u003c/em\u003e, \u003cem\u003eTKN2\u003c/em\u003e, \u003cem\u003eTKN4\u003c/em\u003e, and \u003cem\u003eAPRR2\u003c/em\u003e are factors related to pigments [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] that regulate the production, differentiation, and function of chloroplasts; play important roles in immune and stress responses; and are important nodes in the regulatory network [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. BEL1-LIKE HOMEODOMAIN4 can inhibit the expression of TKN2, which alters the chlorophyll content and chloroplast development in tomatoes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Although research on pigment genes is diverse, studies on the stem color of cucurbit crops are lacking, and candidate genes regulating stem color need to be identified and analyzed.\u003c/p\u003e \u003cp\u003eTherefore, to elucidate the genes regulating stem color in wax gourds, we constructed a high-density genetic map (HDM) of wax gourds using quantitative trait loci sequencing (QTL-seq) and resequencing data of a high-generation inbred line GX-71 (green stem), a high-generation inbred line MY-1 (white stem), and 105 RIL populations (unpublished data).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003ePhenotypic and genetic analyses of the stem color\u003c/h2\u003e\n\u003cp\u003eThe stem colors of the parents, GX-71 and MY-1, used in this study significantly differed. In the field, GX-71, MY-1, and the F1 generation had green, white, and light green stems, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e); thus, these gourds were selected for constructing populations used in the QTL location mining of stem color-related QTLs and candidate genes. To analyze the heritability of the stem color, an F2 population of 786 individuals was bred from the parents, GX-71 and MY-1. In the F2 population, 449 and 337 strains had green and white stems, respectively. The separation ratio of the F2 population was close to 9:7 (x\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.244, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.197), indicating that stem color traits may be regulated by the two major effect genes (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eDistribution of stem color in wax gourd populations\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePlant type\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTotal no.\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGreen\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eWhite\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eX\u003csup\u003e2\u003c/sup\u003e (9:7)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eP\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGX-71\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMY-1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF1 population\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eF2 population\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e786\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e449\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e337\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.244\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.19\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eDetermination of pigment content\u003c/h2\u003e\n\u003cp\u003eThe chlorophyll and carotenoid contents of the parental stems at different developmental stages were measured at 3, 6, 10, 15, 20, 25, 30, and 40 d after transplanting. At 15 d post-transplant, the chlorophyll content of the green-stemmed parent GX-71 gradually increased, whereas that of the white-stemmed parent MY-1 did not change significantly (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). At 40 d post-transplant, the chlorophyll content of GX-71 was more than five times that of MY-1, and the carotenoid content of GX-71 was also significantly higher than that of MY-1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eQTL mapping and candidate gene identification\u003c/h2\u003e\n\u003cp\u003eQTL mapping was performed using a high-density genetic linkage map (unpublished data) and phenotypic data. The map contained 128,9176 variation sites and 1,256,985 markers, with a total distance of 1,345.1 cM. Based on the observed phenotypic values of the 105 inbred lines and HDM (unpublished), QTL mapping of stem color-related traits was performed using the CIM method. Two QTLs, qSC5 and qSC12, were identified as the main stable QTLs on 2 chromosomes. Specifically, qSC5 and qSC12 are located on Chr05 (11,134,567\u0026ndash;16,459,268) and Chr12 (74,618,168\u0026ndash;75,712,335), respectively. The explainable phenotypic variation rate and maximum LOD of qSC5 were 36.9% and 16.9, respectively, while those of qSC12 were 20.9% and 11.2, respectively. Based on the combination of QTL results with unpublished data of the wax gourd reference genome (GX-19) with the GX-71 and MY-1 resequencing data, eight pairs of polymorphic InDel markers were developed for genotyping and linkage analysis of qSC5. Finally, the interval, which contained 14 genes, was narrowed to js13.2 (13,205,354) and js13.8 (13,838,411), with a total length of 633.057 kb (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). In the constructed F2 population, individuals with different stem color traits without qSC5 exchange were selected for self-pollination, and an F2-1 population of 1,120 individuals was obtained. Four pairs of polymorphic InDel markers were developed to narrow qSC12 to a 569.389 kb region containing only 13 genes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Gene sequence and annotation analyses(Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) of the genes in this region showed that \u003cem\u003eBch05G003950\u003c/em\u003e exhibited two-base deletions in the maternal coding sequence, resulting in the formation of a premature termination codon (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Additionally, the gene annotation of \u003cem\u003eBch12G020400\u003c/em\u003e indicated that it encodes phospholipase A (1) DAD1, a protein with chloroplast-like characteristics (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eGenes within the narrowed qSC5 interval\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGene ID\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNonsynonymous Mutations in Coding Sequences\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePhysical Location\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGene Annotation\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch05G003860\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr5: 13207272\u0026ndash;13212788 (+)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eprotein NRT1/ PTR FAMILY 7.3-like\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch05G003870\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr5: 13213544\u0026ndash;13235133 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePyruvate kinase isozyme G\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch05G003880\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr5: 13274698\u0026ndash;13276057(+)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch05G003890\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr5: 13276096\u0026ndash;13281871(+)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProtein ABCI7, chloroplastic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch05G003900\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr5: 13341180\u0026ndash;13346696(-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProbable ribose-phosphate Pyrophosphokinase 1-like\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch05G003910\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr5: 13367114\u0026ndash;13372139(-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProbable tobamovirus multiplication protein 2B isoform X1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch05G003920\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr5: 13438283\u0026ndash;13441573 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProbable peroxidase 20 isoform X1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch05G003930\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr5: 13441626\u0026ndash;13447889 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTobamovirus multiplication protein 2A-like\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch05G003940\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr5: 13484454\u0026ndash;13484678 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch05G003950\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr5: 13491669\u0026ndash;13499644 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTwo-component response Regulator-like protein APRR2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch05G003960\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr5: 13676056\u0026ndash;13689823 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMSC domain-containing protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch05G003970\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr5: 13805666\u0026ndash;13806021 (+)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eVacuolar protein sorting-associated protein 55 homolog\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch05G003980\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr5: 13810161\u0026ndash;13810902 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eUncharacterized protein LOC111455443\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch05G003990\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr5: 13825779\u0026ndash;13838208 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTranscription factor MAMYB\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eGenes within the narrowed qSC12 interval\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGene ID\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNonsynonymous Mutations in Coding Sequences\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePhysical Location\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGene Annotation\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch12G020370\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr12: 75155250\u0026ndash;75160607 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProbable H/ACA ribonucleoprotein complex subunit 1-like\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch12G020380\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr12: 75215164\u0026ndash;75221452 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRGG repeats nuclear RNA binding protein A-like isoform X2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch12G020390\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr12: 75225625\u0026ndash;75297961 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProbable glycine\u0026ndash;tRNA ligase, chloroplastic/mitochondrial 2 isoform X1-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch12G020400\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr12: 75332840\u0026ndash;75335150 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePhospholipase A(1) DAD1, chloroplast-like\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch12G020410\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr12: 75409841\u0026ndash;75418358 (+)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCarotenoid cleavage dioxygenase 7, chloroplastic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch12G020420\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr12: 75418509\u0026ndash;75419660 (+)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProbable major pollen allergen Ole e 6-like\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch12G020430\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr12: 75498816\u0026ndash;75499589 (+)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProbable major pollen allergen Ole e 6-like\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch12G020440\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr12: 75510818\u0026ndash;75511148 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch12G020450\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr12: 75522841\u0026ndash;75523897 (+)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProbable major pollen allergen Ole e 6-like\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch12G020460\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr12: 75529773\u0026ndash;75530669 (+)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProbable major pollen allergen Ole e 6-like\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch12G020470\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr12: 75548538\u0026ndash;75551483 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch12G020480\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr12: 75557730\u0026ndash;75559794 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePentatricopeptide repeat-containing protein At4g01570\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBch12G020490\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eChr12: 75564027\u0026ndash;75565069 (-)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProbable uncharacterized protein LOC103498828\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eqRT-PCR analysis\u003c/h2\u003e\n\u003cp\u003eTo further analyze the candidate genes, qRT-PCR was performed to examine the expression of genes within the parental region. Stem segments with stable stem color traits were selected from the transplanted 40DAT as materials for differential expression analysis of all genes in the region. Differential expression analysis showed that the expression of only \u003cem\u003eBch05G003950\u003c/em\u003e and \u003cem\u003eBch12G020400\u003c/em\u003e differed significantly when compared with those of their parent plants. First, \u003cem\u003eBch05G003950\u003c/em\u003e exhibited increased relative expression in the parental plant GX-71 and reduced relative expression in the maternal plant MY-1 (Fig.\u0026nbsp;4A). Second, \u003cem\u003eBch12G020400\u003c/em\u003e expression was negligible in the maternal plant, but that in the parental plant exhibited significant variation (Fig.\u0026nbsp;4B). These results indicate that \u003cem\u003eBch05G003950\u003c/em\u003e and \u003cem\u003eBch12G020400\u003c/em\u003e may be key genes regulating the stem color in wax gourds.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eInDel marker-assisted breeding\u003c/h2\u003e\n\u003cp\u003eAn InDel marker tightly linked to \u003cem\u003eBchAPRR2\u003c/em\u003e was developed to verify the consistency of the stem color genotypes and phenotypes of the experimental wax gourds. In total, 55 wax gourd germplasm resources with extreme stem color traits were selected to verify the InDel marker. Among them, 34 and 21 had green and white stems, respectively. The results showed that 36 and 19 germplasm resources were consistent with green-stem male parent GX-71 bands and white-stem female parent bands, respectively(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The coincidence rate of the genotype and phenotype was 81.8%. (Supplementary Table\u0026nbsp;2).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe stem color of wax gourds is of great significance for their development and fruiting. Differences in chlorophyll content in the stems of wax gourds lead to different stem colors. Numerous studies have been carried out on pigments, but the stem color in Cucurbitaceae has been sparsely investigated. These studies have shown that pigment type and content can determine the color of plant tissues [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and that main pigment types can differ by tissue. In some plant tissues, such as the peel of eggplant, chlorophyll and anthocyanins are the primary pigments that determine color [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In Chinese kale, which has purple stems, anthocyanin is the main pigment that governs the stem color; the \u003cem\u003eBoDRF\u003c/em\u003e gene, which controls the purple stem color of Chinese kale, is located at a 0.32 cM interval. However, the insertion of a base in this gene leads to a frameshift mutation, resulting in the production of green stems [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Chlorophyll is the main component of green tissues; therefore, a lack of chlorophyll in green tissues leads to plant albinism [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, the main pigment controlling the stem color of wax gourds was determined to be chlorophyll; thus, we observed and measured chlorophyll changes in wax gourd plants for forty days post-transplantation. During this period, the stem chlorophyll contents of parental GX-71 and maternal MY-1 plants significantly differed.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMolecular mechanism regulating the stem color of wax gourds\u003c/h2\u003e \u003cp\u003eVarious family factors, including the GLKs, MYB, APRR, and other families, are reportedly involved in the regulation of pigment traits. For example, in strawberries, FaMYB controls the formation of red strawberries by regulating flavonoid biosynthesis during the late stage of fruit development. Wang et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] inserted eight bases in a specific variant allele of \u003cem\u003eFaMYB10\u003c/em\u003e, which resulted in the formation of a premature termination codon and production of white octoploid strawberries. Additionally, the GLK family transcription factors, which are key nodes in the plant regulatory network, trigger the expression of photosynthesis-related nuclear genes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. For example, the chlorophyll regulatory factor BGP4 is reportedly affects chlorophyll contents by influencing in the conduction of light signals, interaction with the GLK transcription factor, and inhibition of activity of the GLK transcription factor [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In this study, the main regulatory gene for wax gourd stem color was found to be \u003cem\u003eBchAPRR2\u003c/em\u003e, which belongs to the APRR family. In squash, deletion of the chromosomal fragment of the repeated locus on APRR2 leads to the synthesis of chlorophyll in stems, which affects the production of white-stem varieties of squash [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moreover, APRR1 is related to pigments [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and Nong et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] reported that APRR2 is closely related to the skin color of wax gourds. Furthermore, allele variation in \u003cem\u003eBchAPRR2\u003c/em\u003e across varieties of wax gourds affects the plant chlorophyll content and structure. Moreover, APRR family genes are reportedly involved in the regulation of circadian rhythms in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The \u003cem\u003eAPRR9\u003c/em\u003e gene of the APRR family encodes regulatory factors related to light-induced photoresponses and participates in the regulation of the circadian rhythm in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this study, annotation of the \u003cem\u003eBch12G020400\u003c/em\u003e gene revealed that it encodes phospholipase A(1) DAD1, a chloroplast-like protein. In \u003cem\u003eArabidopsis\u003c/em\u003e, the DAD1 protein is chloroplast phospholipase A1, which is a fluorescent green fusion protein mainly located in chloroplasts [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, high expression of \u003cem\u003eBch12G020400\u003c/em\u003e may promote the production of the green fluorescent protein DAD1, thereby affecting the stem color of wax gourds.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we observed that two-base deletions of the main regulatory gene, \u003cem\u003eAPRR2\u003c/em\u003e, of stem color led to a frameshift mutation, which may change the structure and function of the protein. High expression of the secondary regulatory gene \u003cem\u003eBch12G020400\u003c/em\u003e may promote the production of the green fluorescent protein DAD1 in the chloroplast. \u003cem\u003eBchAPRR2\u003c/em\u003e and \u003cem\u003eBch12G020400\u003c/em\u003e may affect the synthesis, structure, or function of proteins in chloroplasts via complex molecular regulatory mechanisms, ultimately leading to stem color differences in wax gourds. Two genes, \u003cem\u003eBchAPRR2\u003c/em\u003e and \u003cem\u003eBch12G020400\u003c/em\u003e, were found to be related to the stem color trait of wax gourd. However, further research on the molecular mechanisms of \u003cem\u003eBchAPRR2\u003c/em\u003e and \u003cem\u003eBch12G020400\u003c/em\u003e regulating wax gourd stem color may face some difficulties, mainly due to the incomplete establishment of the wax gourd genetic transformation system.Our study provides a theoretical basis for further research on these mechanisms and a reference for stem color regulatory genes of other gourd species, and our findings will help genetically improve wax gourd stem color and specific germplasm resources. Further validation of the molecular mechanisms of \u003cem\u003eBchAPRR2\u003c/em\u003e and \u003cem\u003eBch12G020400\u003c/em\u003e in regulating wax gourd stem color is needed and can be achieved by combining genetic transformation methods, gene editing, and overexpression techniques.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePlant material and phenotypic evaluation\u003c/h2\u003e \u003cp\u003eIn this study, 105 recombinant inbred gourd lines were constructed. Green-stemmed GX-71 (male parent) and white-stemmed MY-1 (female parent) plants were utilized as parents to produce populations for QTL localization. The F1 generation was hybridized, and then the F1 generation individuals were self-pollinated to obtain F2 population, comprising 2,218 plants. Individuals with different stem colors from F2 without qSC5 exchange were selected for self-pollination to obtain the F2-1 line. F2 was used for fine-mapping of genes in qSC5, and F2-1 was used for fine-mapping of genes in qSC12. Simultaneously, 55 germplasm resources were used to verify the InDel markers developed in this study. All materials were procured from Nanning Kenong Seed Industry Co., Ltd., China, and planted at the Nanning Yong'an Wax Gourd Experimental Base (E-108\u0026deg;51', N 22\u0026deg;48') from July 2022 to July 2023 for growth under natural light. The parents of the hybrid combination and F2 generation plants were labeled with serial numbers. The wax gourd plants used for phenotypic determination were collected 40 d after transplantation, which ensured that chlorophyll was in a stable state. The stem color of wax gourds was visually evaluated in the field and categorized as white and green. Specifically, a stem color resembling the white stem of the mother plant was recorded as white, and a color resembling the green stem of the father plant was recorded as green.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExtraction and determination of chlorophyll and carotenoid contents\u003c/h2\u003e \u003cp\u003eStems were collected at the same plant height at 0, 3, 6, 10, 15, 20, 25, 30, and 40 d after transplanting. The stems were cut into ~\u0026thinsp;3 cm-long segments using a knife and ground into a powder with liquid nitrogen. Powder was weighed (1.0 g), placed into a 15 mL centrifuge tube, and shaken in a light-proof environment at 200 rpm for 12 h to extract pigments. The chlorophyll a, chlorophyll b, and carotenoids contents were evaluated at 665, 649, and 470 nm, respectively. The respective equation was derived from Li et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] (Appendix).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction\u003c/h2\u003e \u003cp\u003eA plant genomic DNA extraction kit (Solarbio Science, Beijing, China) was used to extract genomic DNA from the young leaves. The obtained DNA was quantified using an ultra-micro spectrophotometer (K5800, KAIAO, Beijing, China) and its integrity was evaluated using 1.2% agarose gel electrophoresis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eQTL mapping and InDel marker analysis\u003c/h2\u003e \u003cp\u003eQTL mapping was performed using a high-density genetic linkage map (unpublished data) and phenotypic data. The map contained 1,289,176 variation sites, 1,256,985 markers, and had a total distance of 1,345.1 cM. QTL mapping was performed using the composite interval mapping (CIM) method and QTL Cartographer (version 1.17j) software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://brcwebportal.cos.ncsu.edu/qtlcart/\u003c/span\u003e\u003cspan address=\"https://brcwebportal.cos.ncsu.edu/qtlcart/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for preliminary localization. InDel markers were developed for genotyping and linkage analysis using Premier 5.0 software based on the whole-genome resequencing data of the parents and 105 inbred lines. Several developed InDel polymorphic molecular markers were used in combination with the F2 population data for fine-mapping of genes in qSC5. Other developed InDel polymorphic molecular markers were used in combination with F2-1 data for fine-mapping of genes in qSC12.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and candidate gene prediction analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from GX-7 and MY-1 using the EastepSuper Total RNA Extraction Kit (Promega, Beijing, China) according to the manufacturer's instructions. Gene sequence comparisons were performed between the candidate gene sequences, which were obtained via the resequencing of the parents and 105 inbred lines. The corresponding gene annotations were searched in the existing wax gourd reference genome, GX-19. Gene sequence alignments were produced using DNAMAN v.9 software (Lynnon Biosoft, San Ramon, CA, United States). Finally, gene sequence analyses combined with gene annotation analysis were used to screen candidate genes involved in the regulation of wax gourd stem color.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eqRT-PCR analysis of candidate genes\u003c/h2\u003e \u003cp\u003eIn this study, qRT-PCR was used to quantify the differential expression of candidate genes in the parent plants. First, total RNA was extracted from parental stem segments 40 d after transplantation and reverse-transcribed using a reverse transcriptase RT Master Mix (Takara, Beijing, China). Primer sequences to amplify the reference genes CAC (\u003cem\u003eBch05G003650\u003c/em\u003e), \u003cem\u003eBch05G003950\u003c/em\u003e, and \u003cem\u003eBch12G020400\u003c/em\u003e and candidate genes were designed using Premier 5.0 (Supplementary Table\u0026nbsp;1). The qPCR analysis was performed using a premixed SYBR Green quantitative PCR reagent and an Applied Biosystems 7500 qRT-PCR system (Foster City, CA, USA). Each experiment was repeated three times, and relative expressions were determined using the 2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e method [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Differences in relative expression were analyzed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMolecular marker-assisted breeding\u003c/h2\u003e \u003cp\u003eA pair of InDel markers tightly linked to the predicted main regulatory gene \u003cem\u003eBchAPRR2\u003c/em\u003e was designed using Premier 5.0. The primer sequences are listed in Supplementary Table\u0026nbsp;1. Combined with developed markers, 55 wax gourd germplasms from the parent plants and F1 with extreme stem color traits, including 34 green stems and 21 white stems, were used for molecular marker-assisted and stem color accuracy verification experiments (Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Guangxi Science and Technology Base \u0026amp; Talents Fund (Guike AD21220040), National Natural Science Foundation of China (31960593), and the Science and Technology Major Project of Guangxi (Guike AB21220029).\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eZC: Data curation, validation, writing \u0026ndash; original draft, formal analysis. PW: methodology, writing \u0026ndash; review \u0026amp; editing. WB: Writing \u0026ndash; review \u0026amp; editing, data curation, investigation. YD: Data curation, investigation, writing \u0026ndash; review \u0026amp; editing. ZKC: Investigation, writing \u0026ndash; review \u0026amp; editing, software. LS: Investigation, writing \u0026ndash; review \u0026amp; editing. LN: investigation, writing \u0026ndash; review \u0026amp; editing. TL: Investigation, writing \u0026ndash; review\u0026amp; editing. WY: Investigation, writing \u0026ndash; review \u0026amp; editing. XY: Investigation, writing \u0026ndash; review \u0026amp; editing, methodology. ZL: Writing \u0026ndash; review \u0026amp; editing, methodology, conceptualization.\u003c/p\u003e\n\u003cp\u003eData Availability\u003c/p\u003e\n\u003cp\u003eThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: CNSA accession number:CNP0004715,https://db.cngb.org/search/?q=CNP0004715.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe are grateful to Guangxi University for providing the experimental instruments for this study.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWu W, Wang P, Huang X, Su L, Lv H, Gou J, Cheng Z, Ma L, Yu W, Liu Z. Fine Mapping and Functional Analysis of Major Regulatory Genes of Soluble Solids Content in Wax Gourd (\u003cem\u003eBenincasa Hispida\u003c/em\u003e). 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GLK Transcription Factors Accompany ELONGATED HYPOCOTYL5 to Orchestrate Light-induced Seedling Development in \u003cem\u003eArabidopsis\u003c/em\u003e. Plant Physiol. 2024;kiae002. https://doi.org/10.1093/plphys/kiae002.\u003c/li\u003e\n\u003cli\u003eTachibana R, Abe S, Marugami M, Yamagami A, Akema R, Ohashi T, Nishida K, Nosaki S, Miyakawa T, Tanokura M, Kim JM, Seki M, Inaba T, Matsui M, Ifuku K, Kushiro T, Asami T, Nakano T. BPG4 Regulates Chloroplast Development and Homeostasis by Suppressing GLK Transcription Factors and Involving Light and Brassinosteroid Signaling. Nat Commun. 2024;15:370. https://doi.org/10.1038/s41467-023-44492-5.\u003c/li\u003e\n\u003cli\u003ePan Y, Bradley G, Pyke K, Ball G, Lu C, Fray R, Marshall A, Jayasuta S, Baxter C, Wijk RV, Boyden L, Cade R, Chapman NH, Fraser PD, Hodgman C, Seymour GB. Network Inference Analysis Identifies an \u003cem\u003eAPRR2-Like\u003c/em\u003e Gene Linked to Pigment Accumulation in Tomato and Pepper Fruits. Plant Physiol. 2013;161(3):1476\u0026ndash;85. https://doi.org/10.1104/pp.112.212654.\u003c/li\u003e\n\u003cli\u003eNong L, Wang P, Yang W, Liu T, Su L, Cheng Z, Bai W, Deng Y, Chen Z, Liu Z. Analysis of \u003cem\u003eBhAPRR2\u003c/em\u003e allele variation, chlorophyll content, and chloroplast structure of different peel colour varieties of wax gourd (\u003cem\u003eBenincasa hispida\u003c/em\u003e) and development of molecular markers. Euphytica. 2023;219:107. https://doi.org/10.1007/s10681-023-03233-x.\u003c/li\u003e\n\u003cli\u003eMatsushika A, Makino S, Masaya K, Mizuno T. Circadian Waves of Expression of the APRR1/TOC1 Family of Pseudo-Response Regulators in \u003cem\u003eArabidopsis Thaliana\u003c/em\u003e: Insight into the Plant Circadian Clock. Plant Cell Physiol\u003cem\u003e. \u003c/em\u003e2000;41(9):1002\u0026ndash;12. https://doi.org/10.1093/pcp/pcd043.\u003c/li\u003e\n\u003cli\u003eIto S, Nakamichi N, Matsushika A, Fujimori T, Yamashino T, Mizuno T. Molecular Dissection of the Promoter of the Light-induced and Circadian-controlled \u003cem\u003eAPRR9\u003c/em\u003e Gene Encoding a Clock-associated Component of \u003cem\u003eArabidopsis Thaliana\u003c/em\u003e. Biosci Biotech Bioch. 2005;69(2):382\u0026ndash;90. https://doi.org/10.1271/bbb.69.382.\u003c/li\u003e\n\u003cli\u003eIshiguro S, Kawai-Oda A, Ueda J, Nishida I, Okada K. The \u003cem\u003eDEFECTIVE IN ANTHER DEHISCENCE1\u003c/em\u003e Gene Encodes a Novel Phospholipase A1 Catalyzing the Initial Step of Jasmonic Acid Biosynthesis, Which Synchronizes Pollen Maturation, Anther Dehiscence, and Flower Opening in \u003cem\u003eArabidopsis\u003c/em\u003e. Plant Cell. 2001;13(10):2191\u0026ndash;209. https://doi.org/10.1105/tpc.010192.\u003c/li\u003e\n\u003cli\u003eLi H. Principles and Techniques of Plant Physiological and Biochemical Experiments. 1st ed. Beijing: Higher Education Press; 2000.\u003c/li\u003e\n\u003cli\u003eLivak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e\u0026Delta;\u0026Delta;\u003cem\u003eC\u003c/em\u003e\u003c/sup\u003e\u003csub\u003eT\u003c/sub\u003e Method. Methods.\u003cem\u003e \u003c/em\u003e2001;25:402\u0026ndash;8. https://doi.org/10.1006/meth.2001.1262.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"wax gourd, regulatory gene, stem color, gene mapping, sequencing, chlorophyll","lastPublishedDoi":"10.21203/rs.3.rs-4134687/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4134687/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eOwing to its nutritional and health benefits, wax gourd [\u003cem\u003eBenincasa hispida\u003c/em\u003e (Thunb) Cogn. (2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;24)] is a staple vegetable variety in China, especially southern China [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Stem color is an important agronomic trait of wax gourds; however, its regulatory genes have not been identified.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this study, 105 inbred lines constructed from two parents (GX-71 and MY-1) were sequenced again, and quantitative trait loci sequencing (QTL-seq) was used to mine the genes that regulate stem color in wax gourds.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTwo QTLs related to stem color, qSC5 and qSC12, were identified. QTL localization revealed, for the first time, that the stem color QTL qSC5 and qSC12 are located on Chr05 (11,134,567\u0026ndash;16,459,268) and Chr12 (74,618,168\u0026ndash;75,712,335), respectively. The explainable phenotypic variation rate and maximum limit of detection(LOD)of qSC5 were 36.9% and 16.9, respectively, while those of qSC12 were 20.9% and 11.2, respectively. Additionally, \u003cem\u003eBch05G003950\u003c/em\u003e (named BchAPRR2) and \u003cem\u003eBch12G020400\u003c/em\u003e were identified as candidate genes involved in stem color regulation in wax gourds. Moreover, the chlorophyll content and fluorescence expression levels of \u003cem\u003eBchAPRR2\u003c/em\u003e and \u003cem\u003eBch12G020400\u003c/em\u003e were significantly higher in green-stemmed wax gourds than those in white-stemmed ones. Therefore, \u003cem\u003eBchAPRR2\u003c/em\u003e and \u003cem\u003eBch12G020400\u003c/em\u003e were considered the main and secondary regulatory genes for wax gourd stem color, respectively. Finally, InDel markers closely linked to BchAPRR2 were developed to validate the prediction of wax gourd stem color traits in 55 germplasm lines, with an accuracy of 81.8%.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study identified the main and secondary genes regulating stem color in wax gourds; these findings lay the foundation for exploring the genetic regulation of wax gourd stem color and future research on wax gourd breeding.\u003c/p\u003e","manuscriptTitle":"Quantitative trait loci sequencing and genetic mapping reveal two main regulatory genes for stem color in wax gourds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-27 19:58:46","doi":"10.21203/rs.3.rs-4134687/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0948b56d-4deb-4305-8d29-4d3f83251e55","owner":[],"postedDate":"March 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-07-08T10:34:53+00:00","versionOfRecord":{"articleIdentity":"rs-4134687","link":"https://doi.org/10.3390/plants13131804","journal":{"identity":"plants","isVorOnly":true,"title":"Plants"},"publishedOn":"2024-06-29 10:34:53","publishedOnDateReadable":"June 29th, 2024"},"versionCreatedAt":"2024-03-27 19:58:46","video":"","vorDoi":"10.3390/plants13131804","vorDoiUrl":"https://doi.org/10.3390/plants13131804","workflowStages":[]},"version":"v1","identity":"rs-4134687","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4134687","identity":"rs-4134687","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}