{"paper_id":"2b5ea917-8cf5-427d-ad74-aa9152dbe7e1","body_text":"Genome-wide investigation of WRKY gene family in Lagenaria siceraria\nYaling Wang1, Qing Lai2, Min Wang2, Hui Zhu2, Dafu Ru2*, Xiaonong Guo1* \n1 School of Life Sciences and Engineering, Northwest Minzu University, Lanzhou730030, China.\n2 State Key Laboratory of Grassland Agro-Ecosystems, and College of Ecology, Lanzhou University, Lanzhou \n730000, China. \n* To whom correspondence should be addressed. Tel. 13880788291; 13679427687. Email: rudf@lzu.edu.cn \n(D.R.); gxnwww@xbmu.edu.cn\nAbstract \nBackground: A significant family of transcription factors known as WRKY genes include many \nphysiological functions and environmental adaptations. However, insufficient information was \npreviously available about the WRKY genes in Lagenaria siceraria, a crucial crop with substantial \neconomic significance. The recent publication of the whole-genome sequence of L. siceraria has allowed \nus to perform a genome-wide investigation of the organization of the WRKY genes in L. siceraria. \nResults: In the present study, 57 L. siceraria WRKY (LsiWRKY) genes were identified and given new \nnames based on their relative chromosomal distribution. The 57 LsiWRKYs were further divided into \nthree major groups and several subgroups based on their structural and phylogenetic properties. \nSegmentation duplication events have played a major role in the expansion of the WRKY gene family in \nL. siceraria. Phylogenetic comparisons of the Group III WRKY genes provide valuable insights into the \nevolutionary characteristics of WRKY genes in L. siceraria. Additionally, RNA-seq analysis revealed \ndistinct expression pattern of WRKY genes across different tissues.\nConclusions: This study presents a preliminary analysis of the WRKY gene family in L. siceraria, \nincluding their structural characteristics, evolutionary traits, and tissue-specific expression patterns. The \nsystematic insights provided here serve as a foundation for further functional studies aimed at enhancing \nL. siceraria crops. This knowledge holds promise for improving the cultivation and yield of L. siceraria, \nthereby contributing to agricultural advancements.\nKeywords: Lagenaria siceraria, WRKY, genome-wide\nBackground\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\nTranscription factors (TFs) are the class of proteins that can interact with other regulatory factors or bind \nto specific DNA sequences in the promoter regions of genes, thereby regulating the functioning of \nvarious genes and thus involved in downstream target genes  regulation process (Franco-Zorrilla et al., \n2014; Li et al., 2015). As an influential form of TFs, the WRKY gene family has been studied for more \nthan two decades since it was first cloned from sweet potato (Ishiguro et al., 1994) and primarily found \nin single-celled algae and plants. They are named as WRKY because the protein sequence contains \nseveral, highly conserved WRKY domains, which include about 60 amino acids. Each and every WRKY \nprotein that has been identified has one or two WRKY domains at the N-terminus, followed by zinc \nfinger motifs at the C-terminus (Li et al., 2015). The classification of WRKY proteins into three broad \nclasses is based on the quantity of WRKY domains and the type of zinc finger sequences (I-III).  \nMembers of group I have two WRKY domains and a zinc-finger motif of the C2H2 type, whereas group \nII and group III only have one WRKY domain and follow it with zinc finger motifs of the C2H2 and \nC2HC types, respectively (Eulgem et al., 2000). The WRKYs of group II can be further classified into \nfive different subgroups based on their phylogenetic relationship (IIa-e). Through the identification of \nthe W-box core sequence (TTGACC/T) within the promoter region of target genes, WRKY transcription \nfactors exhibit exclusive binding to these target genes (Yu et al., 2001).\nAccording to incomplete statistics, more than 14,500 WRKY proteins have been identified from \n165 plant species (Jin et al., 2017). The great majority of WRKY protein research have shown that these \nproteins are engaged in a variety of biological and abiotic stress responses as well as playing important \nroles in the plant immune system. By way of illustration, increasing the expression of AtWRKY4 can \nmake plants more susceptible to the biotrophic bacterium Psudomonas siringae (Lai et al., 2008). In \nCucumis, LsiWRKY50 plays a positive role in Pseudoperonospora cubensis resistance involving multiple \nsignaling pathways (Luan et al., 2019). In comparison to wild-type plants, OsWRKY47 overexpression \ncan boost rice yield and drought resistance (Raineri et al., 2015). In response to salinity stress, \nGmWRKY92, GmWRKY144, and GmWRKY165 would be positively regulated in soybeans (Song et al., \n2016). It has been shown that OsWRKY11 overexpression can increase tolerance to stress caused by high \ntemperatures (Wu et al., 2009). VvWRKY30 was proved to confer tolerance to salt stress in Vitis vinifera \n(Zhu et al., 2019). Moreover, WRKY proteins also participate in additional crucial plant processes, such \nas pollen development (Lei et al., 2017), seed size (Luo et al., 2005; Gu et al., 2017), seed dormancy and \ngermination (Jiang et al., 2009; Xie et al., 2007; Zou et al., 2008), plant development (Johnson et al., \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\n2002; Ishida et al., 2007) and leaf senescence (Ay et al., 2009; Brusslan et al., 2012; Yang et al., 2016). \nNearly all WRKY families in angiosperms have undergone significant expansion during evolution due \nto the substantial involvement of the WRKY family in a variety of physiological activities. For instance, \nthere are at least 70 WRKY proteins in Arabidopsis (Eulgem et al., 2000; Dong et al., 2003), 174 in \nGlycine max (Yang et al., 2017b; Yang et al., 2017a) and 109 in rice (Wu et al., 2005).\nAlthough a large number of studies have been published on the WRKY gene family, relatively few \nhave investigated the bottle gourd. One of the major crops in the Cucurbitaceae, the bottle gourd \n(Lagenaria siceraria), is a diploid species (2n=2x=22), and it possesses a genome size of 313.4 Mb (Wu \net al., 2017). It is believed to have originated in southern Africa and is now widely grown in the tropical \nand subtropical regions (Wu et al., 2017), particularly in the East Asian countries (Kistler et al., 2014). \nDue to its beneficial nutritional properties (Loukou et al., 2011) and health properties (Shah et al., 2010), \nL. siceraria has a significant potential for usage in medicines. For instance, it hydrates the skin and \ndecreases edema and knots. It also can be used for food, containers, decorative artefacts or musical \ninstruments (Mashilo et al., 2017). In order to enhance the cold tolerance and disease resistance of other \ncucurbit crops, bottle gourd has recently emerged as a vital rootstock material for grafting (Davis et al., \n2008; King et al., 2008). For instance, the bottle gourd is the recommended rootstock for watermelon, \none of the most widely grown fruits in the world because it controls soil-borne diseases and has no impact \non fruit quality of the fruit (Davis et al., 2008; Fidan et al., 2016). Therefore, the study of important \nfunctional genes in the bottle gourd has aroused substantial interest from researchers. A thorough analysis \nof the WRKY gene family in L. siceraria would be crucial due to the significance of the WRKY genes in \nmany physiological systems. The recent completion of sequencing of the L. siceraria genome provides \nan opportunity to reveal the organization and evolutionary traits of the L. siceraria WRKY gene family \nat the genome-wide level. In the current work, 57 L. siceraria WRKY genes were discovered, and they \nwere divided into three major groups. Comprehensive analyses including the exon-intron organization, \nmotif composition, gene duplication, chromosome distribution, phylogenetic and synteny analysis were \nalso investigated. Our study provides valuable clues to understand the functional characterization of \nmembers of the WRKY gene family in L. siceraria.\nResults\nIdentification of the WRKY genes in L. siceraria\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\nIn this study, we systematically investigated the WRKY gene family in L. siceraria, one of the largest \nfamilies of plant transcription factors. Initially, 61 putative WRKY genes were identified through \nBLASTP using Arabidopsis thaliana genes as references (Table S1). Subsequently, redundant and \nnon-WRKY domain-containing sequences were removed, resulting in the exclusion of specific \nsequences (Lsi01G000510.1, Lsi05G005250.1, Lsi09G001980.1, Lsi09G010130.1). Ultimately, 57 \nWRKY genes were identified and annotated by validating the presence of WRKY domains using the \nSMART program. The number of identified WRKY genes in L. siceraria (Table S2) was comparable \nto that of other plants, such as Cucumis Melo L. (57 members) (Chen et al., 2021), Cucumis sativus \n(57 members) (Ling et al., 2011) and Capsicum annuum L. (61 members) (Cheng et al. 2016). These \n57 WRKY genes were successfully mapped to chromosomes 1–11, and based on their chromosomal \nlocations, they were systematically renamed as LsiWRKY1 to LsiWRKY57 (Fig. 1; Table S2). \nFurthermore, we investigated additional essential features of the WRKY proteins, including \ntheir protein sequence length, coding sequence (CDS) length, molecular weight (MW), and \nisoelectric point (pI) (Table S3). Among the 57 LsiWRKY proteins, LsiWRKY04 with 119 amino \nacids represented the smallest, whereas LsiWRKY44 with 751 amino acids was the largest protein. \nThe molecular weights of these proteins ranged from 13.125 kDa (LsiWRKY04) to 81.409 kDa \n(LsiWRKY44), indicating significant variability in protein sizes. Additionally, the pI of WRKY \nproteins spanned from 4.64 (LsiWRKY16) to 9.73 (LsiWRKY38), underscoring the diverse \nbiochemical properties within this gene family. This comprehensive analysis provides a detailed \noverview of the structural characteristics of the identified LsiWRKY proteins, setting the stage for \na deeper understanding of their functional roles in L. siceraria. \nMultiple sequence alignment and phylogenetic analysis \nIn our investigation, we conducted a multiple protein sequence alignment of all 57 LsiWRKY \nproteins using Muscle software to explore their evolutionary relationships (Fig. S1). Subsequently, \nusing MEGAX software with the neighbour-joining method, a phylogenetic tree was constructed \nbased on the highly conserved WRKY domains of 57 LsiWRKYs and 71 AtWRKYs (Fig. 2). The \nphylogenetic analysis revealed that the 57 LsiWRKYs could be categorized into three major groups \nanalogous to the grouping in Arabidopsis as defined by Eulgem et al. (2000) (Fig. 2). Specifically, \n8 LsiWRKY proteins were classified into group I, 39 into group II, and 7 into group III, while 3 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\nremained unclassified (Fig. 3). In group I, the 8 members shared C2H2-type zinc-finger motifs (C-\nX4-C-X22–23-H-X-H) and possessed both N-terminal and C-terminal WRKY domains. Group II, \ncomprising the majority of LsiWRKYs, was further divided into five subgroups (IIa-IIe). These \nsubgroups exhibited variations in the C2H2-type zinc-finger motifs and contained different numbers \nof WRKY domains (4 WRKY proteins belonged to IIa, 5 to IIb, 17 to IIc, 7 to IId, and 6 to IIe). \nNotably, subgroup IIc was the most abundant, mirroring the grouping pattern observed in \nAtWRKYs. Group III, distinctive due to the zinc-finger motif C2HC: C-X-C-X23-H-X-C, \ncomprised 7 LsiWRKY members, each possessing one WRKY domain. \nInterestingly, our analysis identified a potential R-protein WRKY, LsiWRKY34 from group \nIIc tightly clustered with AtWRKY19, characterized by the presence of the 'leucine-rich repeat' \n(LRR) motif, commonly found in resistance (R) proteins (Fig. 3). This suggests a putative role for \nLsiWRKY34 in L. siceraria's response to biotic or abiotic stresses, akin to the known R-protein \nWRKYs in Arabidopsis (Rinerson et al., 2015; Lobo et al., 2017).\nFurthermore, we scrutinized the conserved domain \"WRKYGQK\", a hallmark of WRKY \ntranscription factors, based on the grouping information. While most LsiWRKYs exhibited the \nWRKYGQK variant, indicating Q to K substitutions, three LsiWRKYs (LsiWRKY04, \nLsiWRKY52, LsiWRKY46) in subgroup IIc displayed WRKYGKK variants (Fig. S1). \nAdditionally, LsiWRKY54 from group I exhibited the mutant WYMRCQM sequence (Fig. S1). \nThese variations, observed mainly in subgroup IIc, mirrored findings in other plant species like \npeanuts and soybeans, highlighting the sensitivity of WRKY domains in this subgroup to mutations \n(Song et al., 2016). Furthermore, our analysis revealed certain WRKY domains with substantial \nsequence variation, leading to their classification into an unclassified group. The origin of these \nvariations could be attributed to potential issues in genomic sequencing or gene prediction programs, \nwarranting further investigation. Overall, these findings shed light on the diversity within the \nLsiWRKY gene family, providing valuable insights into their evolutionary patterns and functional \nsignificance. \nGene structure and motif composition of L. siceraria WRKY gene family\nThe MEME (Multiple EM for Motif Elicitation) tool was employed to reveal the conserved motifs \namong 57 LsiWRKY proteins in order to more fully characterize the structure of the WRKY \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\ndomains. In total, ten conserved motifs, labeled as Motifs 1 through 10, were identified (Fig. 4). \nThese motifs varied in width, spanning 21 to 44 amino acids residues, and were represented by \ndistinct colored boxes (Fig. 4). Among these motifs, Motifs 1 and 3 encoded the conserved WRKY \ndomain, while Motifs 2 and 4 encoded the conserved zinc finger structure. All LsiWRKY proteins \nhave one or two WRKY motifs (Fig. 5). Additionally, conserved motifs (Motifs 4–10) were \nidentified in various LsiWRKY proteins. Furthermore, motif 1 was prevalent in almost all \nLsiWRKYs except for three unclassified genes and LsiWRKY04, LsiWRKY05, LsiWRKY30, \nLsiWRKY52 (Fig. 5). Each LsiWRKY protein harbored at least two conserved motifs, and some \ncontained as many as six motifs (Fig. 5). \nDistinct distributions of conserved motifs were observed across different LsiWRKY groupings. \nFor example, group I LsiWRKYs exhibited 3 to 6 motifs (Motifs 1, 2, 3, 4, and 5), with each member \nin this group possessing at least one of Motifs 1 and 3, as well as at least one of Motifs 2 and 4 (Fig. \n5). Notably, Motif 4 was unique to the group I LsiWRKY proteins (Fig. 5). Motif 9 was exclusively \npresent in Group III and subgroup IId, while Group I and subgroups IIb and IIc contained either \nMotif 4 or Motif 8 (Fig. 5). Subgroups IIa and IIb predominantly featured Motif 7 (Fig. 5). The \nconservation of motif types within the same group indicated similar functionalities among members. \nStudies have shown that the intron-exon structure of multiple gene families plays an important \nrole in plant evolution (Wang et al., 2023; Safder et al., 2021). In order to further understand the \nstructural features of the WRKY family in L. siceraria, we investigated the exon-intron structures of \nidentified LsiWRKY gene. It is obviously that all LsiWRKY genes exhibit two to six exons, with none \nhaving only one exon (Fig. 5). Generally, genes within the same group share a similar structure, as \nhighlighted in brown for group I members (Fig. 5). Intriguingly, each WRKY domain in LsiWRKY \ngenes possessed an intron, except for specific genes such as LsiWRKY42, 20, 29, 53, 52, 57, 48, 7, \n5, and 4, and 34 (Fig. 5). Intron distributions and phase coincided with the alignment of the \nLsiWRKY genes clusters. V-type intron (phase-0 intron) were found in group IIa and II, while R-\ntype intron (phase-1 intron) akin to those in rice and Arabidopsis, were prevalent in other groups \n(group I, IIc, IId, IIe and III) (Rinerson et al., 2015), with N-terminal WRKY domains of group I \nlacking introns (Fig. 5).\nChromosomal distribution of LsiWRKY genes\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\nBased on the information about the location of the genes on the chromosomes, we determined the \npositional distribution of the 57 LsiWRKYs on 11 chromosomes in the L. siceraria genome. From \nthe outputs of the MEME motif analysis, a schematic representation of the structure of all LsiWRKY \nproteins was constructed. With the exception of Motifs 1 and 2, which are broadly dispersed \nLsiWRKY domains, the distribution of LsiWRKY members within the same group is visualized \nacross all L. siceraria chromosomes (Fig. 1; Fig. 5). We found that the distribution of WRKY genes \non each chromosome was not uniform and dense (Fig. 1). Notably, chromosome 9 harbored only \none LsiWRKY gene, whereas chromosome 5 exhibited the largest number of LsiWRKY genes (n=10), \nwhich accounted for 17.5% of all LsiWRKY genes. In addition, chromosomes 1, 4 and 7 contain the \nsame number of LsiWRKY genes, each hosting 6 LsiWRKY loci. Interestingly, several regions of \nhigh LsiWRKY gene density were found on some chromosomes, including 1, 3, and 5, suggesting \npotential WRKY gene hotspots in the genome. \nExpression of WRKY gene family in different tissues of L. siceraria\nPrevious studies have indicated substantial variation in the expression levels and roles of WRKY \ngenes across different tissue (Wang et al., 2019; Fan et al., 2018). To explore the expression patterns \nof different WRKY genes in L. siceraria tissues, we analyzed 10 sample replicates (three fruits, two \nstems, three leaves and two roots) using R (v 4.2.3). Our analysis revealed diverse expression \npatterns among different WRKY gene groups. \nIn group I, several genes exhibited high expression across all tissues. Notably, LsiWRKY10 \ndisplayed elevated expression levels in both fruits and stems, while LsiWRKY19 and LsiWRKY37 \nexhibited lower expression in leaves (Fig. 6). Subgroup IIa genes, with the exception of LsiWRKY48 \nand LsiWRKY49, were highly expressed in all tissues. Conversely, subgroup IIb genes displayed \nrelatively lower expression in all tissues. Subgroup IIc genes exhibited notable functional \ndifferentiation, resulting in significant expression variations across tissues. Subgroup IId genes, \nexcept for LsiWRKY31, showed high expression levels in all tissues, indicating a limited role in leaf \ndevelopment. Subgroups IIe and IIa demonstrated similar expression patterns, being expressed in \nall tissues. Group III featured high expression of LsiWRKY35 and LsiWRKY11, whereas LsiWRKY20 \nexhibited high expression in roots and stems, with other genes displaying lower expression. Genes \nin the unclassified group exhibited high expression levels in all four tissues, except for LsiWRKY53 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\n(Fig. 6). \nDiscussion\nWRKY genes are a family of transcription factors found in all plant species as they play crucial roles \nin regulating plant development and responses to biotic and abiotic stresses. In this study, we used \na genome-wide search which identified 57 putative WRKY genes in L. siceraria, a plant species of \neconomic importance. Through phylogenetic and structural analyses, we categorized the WRKY \ngenes into three groups with several subgroups on the basis of phylogenies and the basic structure \nof the WRKY domains. Notably, we found that the Group I WRKY proteins in L. siceraria retained \nboth WRKY domains and did not undergo any domain loss events during evolution, in contrast to \nwhat has been observed in other plant species. These findings shed light on the evolutionary history \nof the WRKY gene family in L. siceraria, and provide a basis for further investigations into the \nfunctional diversity and regulation of these genes in response to different environmental stimuli. \nPrevious studies have suggested that N-terminal WRKY domains exhibit weak DNA-binding \nactivity and are more variable during evolution. Among the different subgroups of WRKY, Group \nI, which contains two WRKY domains, is considered to be the most ancient member that occurred \nduring the evolution of WRKY. The WRKYs in subgroup IIa and IIb are believed to have originated \nfrom an algal single WRKY domain or from the other Group I derived lineage (Rinerson et al., \n2015; Waqas et al., 2019). Members of subgroup IIc evolved from WRKYs in subgroup II that \nlacked N-terminal domains. However, the origin of each type of WRKY protein in L. siceraria is \ncurrently unknown. Although WRKY domains are strongly conserved among WRKY proteins, \nLsiWRKY proteins exhibit some degree of structural divergence. The heptapeptide WRKYGQK is \nthe typical domain of the WRKY family, but three variants of this domain, including WRKYGKK, \nhave been identified in several LsiWRKY proteins in subgroup IIc. Similar variants of the WRKY \ndomain have also been found in other plant species (Li et al., 2015; Guo et al., 2014; Yue et al., \n2016; Zou et al., 2016), suggesting that these variants may confer multiple biological functions to \nWRKY gene family (Wu et al., 2017). \nSubgroup IIa contains seven LsiWRKY genes and is phylogenetically closer to subgroup IIb \nthan to subgroups IIc. This classification is supported by the fact that the WRKY domains of \nsubgroup IIa and subgroup IIb maintain a similar consensus structure (Fig. S1). In addition, the \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\nresults of the dual phylogeny (Fig. 2) also showed anomalies compared to the results of the single \nspecies phylogeny (Fig. 3), particularly with regard to the positions of LsiWRKY30, LsiWRKY06, \nand ATWRKY49 which clustered together. LsiWRKY30 should belong to Group Ⅰ, LsiWRKY06 to \nsubgroup Ⅱc, and ATWRKY49 to subgroup Ⅱc (Wang et al., 2014; Chen et al., 2020). Based on \nthe close relationship between LsiWRKY06 and ATWRKY49 and both of which belong to subgroup \nⅡc, it appears that LsiWRKY30 was misclassified as subgroup Ⅱc. This may be due to the \nintroduction of ATWRKY49 in the single-species phylogenetic tree resulting in sequence variation \nin the WRKY structural domain of LsiWRKY30 and LsiWRKY06 that was not evident during the \ntree construction, and the phylogenetic software used was not precise enough. Future studies should \nconduct to further differentiate these sequences, which will help to delineate more precise \nphylogenetic relationships. Moreover, from an overall perspective, the WRKY domain of \nATWRKY49 forms a separate cluster of its own, which indicates a high degree of divergence \nbetween this domain and other members of the subgroup Ⅱc. The differentiation between \nLsiWRKY06 and LsiWRKY30 was also evident, although to a lesser extent. Therefore, the results \nsuggest that subgroup Ⅱc and Group Ⅰ are closely related genetically and eventually diverged into \ntwo different subgroups (Fig. 2). It is hypothesized that ATWRKY49 is closely related to subgroup \nIIc and Group I. Furthermore, the sequence alignment results showed that the WRKY domains of \nLsiWRKY29, LsiWRKY53, and LsiWRKY57 contained a large degree of variation, which placed \nthem in an unclassified subgroup not previously known in the WRKY family (Fig. 3; Fig. 2). It is \nworth noting that gene annotation errors may occur (which are rare) due to problems with genome \nsequencing or gene prediction software, and further validation is needed to confirm the identity and \nfunction of these WRKY gene.\nIn addition, we have identified LsiWRKY34 as a chimeric protein that contains both R-protein \nand WRKY domains. Chimeric proteins with both domains have been reported in other plant species \nand have been implicated in plant defense against diseases and stresses. For example, in barley, \nWRKY1/2 inhibited basal defense, but when the Avra10 effector was present, R protein MLA10 \nand WRKY1/2 interacted in the cell nucleus to suppress the effect of WRKY1/2 on basal defense \nand enhance disease resistance (Shen et al., 2007). Similarly, in Arabidopsis, the ATWRKY genes \nencodes an NBS-LRR-WRKY protein that acts as a chimeric protein, with the WRKY domain \nexhibiting DNA-binding activity (Noutoshi et al., 2005). The presence of R protein-WRKY \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\nchimeric proteins in L. siceraria suggests a possible role in plant resistance to disease or other \nstresses, as has been observed in A. thaliana and barley. However, further investigation is needed \nto determine the putative functions of these chimeric proteins in L. siceraria. Overall, the results of \nthis study have important implications for understanding the environmental resistance in plants and \ncould potentially lead to the development of new strategies for improving plant productivity and \nstress resistance.\nOur RNA-seq expression analysis of LsiWRKY genes revealed their presence in all examined \ntissues, displaying diverse and distinct expression patterns. Generally, most LsiWRKYs  displayed \nrelatively high abundance in stems and roots, whereas expression levels were comparatively lower \nin fruits and leaves. However, specific genes exhibited high expression specifically in fruits and \nleaves. Furthermore, except for the subgroup IIc, where gene expression patterns varied \nsignificantly, genes in other subgroups exhibited similar expression patterns, suggesting potential \nfunctional redundancy. Conversely, LsiWRKYs with diverse expression patterns likely fulfill \ndifferent biological functions in plant growth and development. These results lay the groundwork \nfor in-depth analysis of individual WRKY gene expressions in L. siceraria, shedding light on the \nintricate regulatory mechanisms within this gene family.\nConclusions\nA comprehensive analysis of the WRKY gene family in L. siceraria was performed in the present \nstudy. Fifty-seven full-length WRKY genes were characterized and further classified into three main \ngroups, with strongly similar exon-intron structures and motif compositions within the same groups \nand subgroups. Through phylogenetic comparison of WRKY genes from several different plant \nspecies and tissue-specific expression analysis, we gained valuable clues about the evolutionary \ncharacteristics of L. siceraria WRKY genes. Notably, our identification of a chimeric R-protein-\nWRKY protein in L. siceraria suggests a potential role in disease resistance or stress responses, \nalthough further investigation is warranted to fully elucidate its functional significance. The findings \nprovide a solid foundation for future research, offering promising avenues for enhancing agronomic \ntraits and bolstering environmental resistance in this pivotal crop species. Furthermore, our study \nunderscores the broader importance of the WRKY gene family in plant biology, shedding light on \ntheir specific roles in L. siceraria. Overall, this research not only deepens our knowledge of WRKY \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\ngenes but also opens new doors for harnessing their potential in crop improvement, emphasizing \ntheir vital role in the context of plant biology.\nMethods\nGene identification\nTo identify candidate datasets for LsiWRKYs, the L. siceraria whole genome protein database was \nanalyzed using BLASTP (v 2.14.0+; E-value 1e-10; 71 AtWRKYs protein sequences were used as \na query), where AtWRKYs protein sequences were downloaded from the TAIR database \n(https://www.arabidopsis.org). All candidate LsiWRKY protein sequences were eventually \ncharacterized for structural domains through the SMART plugin (http://smart.embl-heidelberg.de/) \nof the TBtools platform (https://github.com/CJ-Chen/TBtools) to test the WRKY conserved \ndomains for integrity, incomplete sequences were excluded from the dataset, and redundant \nsequences were further manually removed. Finally, the Expasy ProtParam tool \n(http://us.expasy.org/tools/protparam.html) was used to calculate the biophysical properties of the \nLsiWRKYs protein sequences, including sequence length, molecular weight and protein isoelectric \npoint.\nGene structure analysis and chromosome Location\nA schematic diagram of the exon-intron organization of L. siceraria WRKY genes was constructed \nusing the online Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/; accessed time \n2023.08.30), in which exon position information was obtained from the genome's annotated ‘gff’ \nfile. After further information on the distribution of LsiWRKY genes on chromosomes was obtained \nfrom the genome annotation files, TBtools was utilized to show the distribution of their positions \non 11 chromosomes.\nMultiple sequence alignment and MEME analysis\nMultiple WRKY protein sequences of L. siceraria were first aligned using MUSCLE (v 5.1) with \ndefault parameters, and the final results of the alignment were visualized using the Genedoc (v 2.7) \nsoftware and presented in Fig S1. Next, to identify conserved motifs in L. siceraria proteins, the \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\nonline MEME program (Multiple Expectation Maximization for Motif Elicitation; http://meme-\nsuite.org/tools/meme/2023.08.30) was used to identify conserved motifs in the 57 identified L. \nsiceraria WRKY protein sequences. The maximum value of the basic search was set to 10, and the \noptimum width of each motif was limited to 21-44 amino acid residues.\nPhylogenetic analysis\nTo elucidate the evolutionary relationships within the WRKY gene family of L. siceraria, a robust \nmethodology was employed. Firstly, multi-protein sequence alignments were performed between \nA. thaliana and L. siceraria and within L. siceraria itself. Subsequently, phylogenetic trees were \nconstructed using both neighbor-joining (NJ) and maximum likelihood (ML) algorithms in the \nMEGA X software. \nFor the NJ analysis, the Dayhoff substitution matrix (PAM250) was used, and the reliability of \nthe constructed trees was verified through 1000 bootstrap replicates, ensuring robustness and \naccuracy in the inferred relationships. \nIn the construction of the ML tree, the best-fit model (JTT+G) was determined from 59 amino \nacid substitution models using the modelfinder tool in MEGA X. This careful model selection \nprocess ensured the appropriateness of the chosen model for the dataset. Following model selection, \nprotein sequence information was integrated, leading to the development of a tree for L. siceraria’s \nWRKY proteins, utilizing the ML algorithm. The resulting ML tree was visually represented using \nthe iTOL tool (https://itol.embl.de/), enhancing the clarity and accessibility of the evolutionary \ninsights. Additionally, the classification of the LsiWRKY gene family was meticulously conducted \nbased on the results derived from the phylogenetic tree analysis and the identification of conserved \ndomains. This rigorous approach ensured a comprehensive understanding of the evolutionary \ndynamics and structural features within the WRKY gene family of L. siceraria. \nTranscriptome data analyses\nTranscriptomic data, comprising three leaf tissues, three fruit seeds, two root tissues, and two stem \ntissues, were downloaded from National Center for Biotechnology Information (NCBI, \nhttps://www.ncbi.nlm.nih.gov/). A total of ten transcriptomic data were included in the analysis. \nPrior to analysis, the data underwent rigorous filtering using trim_galore (v 1.18; \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\nhttps://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Clean reads were then mapped \nto the L. siceraria genome using HISAT2 (v 2.2.1). The resultant data were assembled using \nfeatureCounts (v 2.0.6). For quantification, the expression value of each gene was measured in \nTranscripts Per Kilobase per Million mapped reads (TPM) and calculated using the R (v 4.2.3). R \npackage Heatmap was employed to create an expression level heatmap for different tissues based \non log2(TPM+1) data.\nAcknowledgements\nThis study was supported by Northwest Minzu University Talent Introduction Project \n(Z2101707), the National Natural Science Foundation of China (grant no. 32001085) \nand Fundamental Research Funds for Central Universities (grant no. lzujbky-2020-34).\nSupplementary Material\nFig. 1. Mapping of the WRKY gene family on L. siceraria chromosomes. The size of a \nchromosome is indicated by its relative length. \nFig. 2. The neighbor joining phylogenetic tree of WRKY family genes of Arabidopsis thaliana and \nL. siceraria. Each WRKY group is labeled with different colors.Solid triangles represent L. \nsiceraria and hollow triangles represent Arabidopsis thaliana.\nFig. 3. Phylogenetic tree of LsiWRKY proteins in L. siceraria using the neighbor joining method \nby MEGA X. \nFig. 4. Schematic diagram of conserved motif of WRKY protein in L. siceraria，including the \nmotif logos, consensus sequence widths in aa, and E-values. \nFig. 5. Comprehensive schmatic diagram of phylogenetic clustering,conserved protein motifs, \nLsiWRKYs gene structure. Left panel: the phylogenetic tree was constructed from the WRKY \ndomain sequences of LsiWRKY proteins. Different colors represent different categories. Middle \npanel: the motifs are represented by different colored boxes with corresponding numbers. Right \npanel: gene structure of LsiWRKY. Untranslated 5 ′- and 3 ′-regions, exons, introns, WRKY \ndomains, Plant-zn-clust and WRKY superfamily are indicated by green boxes, yellow boxes, black \nlines, pink boxes, blue-green boxes and red boxes, respectively. Intron phases 0, 1, and 2 are \nindicated by numbers 0, 1 and 2, respectively. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\nFig. 6. The heatmap of expression levels of 57 WRKY genes in L. siceraria.\nFig. S1. Schematic diagram of multiple-sequence alignment of conserved WRKY domains. Top \npanel:the conserved N-terminal LsiWRKY domains of different groups in L. siceraria.And \nhighlight for the variant YMRC sequence. Bottom panel: the conserved C-terminal LsiWRKY \ndomains of different groups in L. siceraria. That belong to the same group are clustered together \nand marked with different colors. The conserved amino acids are highlight with homochromatic \nbackground. \nFig. S2. LsiWRKY proteins domains prediction.\nTable S1. List of the 61 putative WRKY genes were initially identified.\nTable S2. List of the 57 LsiWRKY genes identified in this study.\nTable S3. Physicochemical property of LsiWRKY proteins and grouping.\nTable S4. Results of predicting the conserved domain of LsiWRKY genes using the CD-search tool.\nTable S5. ten transcriptome data and their download sources.\nConflicts of Interest\nThe authors declare that they have no conflicts of interest.\nFunding\nThe author(s) declare that financial support was received for the research and/or \npublication of this article. The work is supported by the Northwest Minzu University \nTalent Introduction Project (Z2101707) and Innovative Fund Project for University \nTeachers in 2024 (2024B-031).\nReferences \nAy, N., et al. Epigenetic programming via histone methylation at WRKY53 controls leaf senescence \nin Arabidopsis thaliana. Plant Journal. 2009; 58:333–346.\nBrusslan, J.A., et al. Genome-wide evaluation of histone methylation changes associated with leaf \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\nsenescence in Arabidopsis. PLoS One. 2012; 7: e33151.\nChen, C., Chen, X., Han, J., Lu, W., Ren, Zet al. Genome-wide analysis of the WRKY gene family \nin the cucumber genome and transcriptome-wide identification of WRKY transcription factors \nthat respond to biotic and abiotic stresses. BMC Plant Biology. 2020; 20:443.\nChen, Y., et al. Genome-wide analysis of WRKY transcription factor family in melon (Cucumis \nMelo L.) and their response to powdery mildew. Plant Mol Biol Rep. 2021; 39, :686–699. \nhttps://doi.org/10.1007/s11105-020-01271-6.\nCheng, Y., et al. In silico identification and characterization of the WRKY gene superfamily in \npepper (Capsicum annuum L.). Genet Mol Res. 2016; 15. doi: 10.4238/gmr.15038675. \nDavis, A.R., et al. Cucurbit grafting Crit. Rev. Plant Sci. 2008; 27: 50-74. doi: \nhttps://doi.org/10.1080/07352680802053940.\nDong, J.X., et al. Expression profiles of the Arabidopsis WRKY gene superfamily during plant \ndefense response. Plant Molecular Biology. 2003; 51:21–37.\nEulgem, T., et al. The WRKY superfamily of plant transcription factors. Trends in Plant Science. \n2000; 5:199–206.\nFan, C.J., et al. Genome-wide analysis of Eucalyptus grandis WRKY genes family and their \nexpression profiling in response to hormone and abiotic stress treatment. Gene. 2018; 678: 38-\n48.\nFidan, H., et al. Open-field survey of Turkish bottle gourd germplasm reaction to virus disease. The \nXIth EUCARPIA Meeting on Genetics and Breeding of Cucurbitaceae. 2016; 283-287 ref.37.\nFranco-Zorrilla, J. M., et al. DNA-binding specificities of plant transcription factors and their \npotential to define target genes. Proc. Natl. Acad. Sci. U. S. A. 2014; 111, :2367–2372.\nGu, Y.Z., et al. Differential expression of a WRKY gene between wild and cultivated soybeans \ncorrelates to seed size. Journal of Experimental Botany. 2017; 68:2717–2729.\nGuo, C., et al. Evolution and expression analysis of the grape (Vitis vinifera L.) WRKY gene family. \nJ. Exp. Bot. 2014; 65:1513–28.\nIshida, T., et al. Arabidopsis TRANSPARENT TESTA GLABRA2 is directly regulated by R2R3 \nMYB transcription factors and is involved in regulation of GLABRA2 transcription in \nepidermal differentiation. Plant Cell. 2007; 19: 2531–2543.\nIshiguro, S., Nakamura, K. Characterization of a cDNA encoding a novel DNAbinding protein, \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\nSPF1, that recognizes SP8 sequences in the 5′ upstream regions of genes coding for sporamin \nand β-amylase from sweet potato. Mol Gen Genet. 1994; 244:563–71.\nJiang, Y., Deyholos, M.K. Functional characterization of Arabidopsis NaCl-inducible WRKY25 \nand WRKY33 transcription factors in abiotic stresses. Plant Mol Biol. 2009; 69: 91–105.\nJin, J., et al. PlantTFDB 4.0: toward a central hub for transcription factors and regulatory \ninteractions in plants. Nucleic Acids Research. 2017; 4: D1040–D10455.\nJohnson, C.S., et al. TRANSPARENT TESTA GLABRA2, a trichome and seed coat development \ngene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell. 2002; 14: 1359–1375.\nKing, S.R., et al. Grafting for disease resistance. HortScience. 2008; 4: 1673– 1676.\nKistler, L., et al. Transoceanic drift and the domestication of African bottle gourds in the Americas. \nProc. Natl Acad. Sci. USA. 2014; 111: 2937– 2941.\nLai, Z., et al. Roles of Arabidopsis WRKY3 and WRKY4 transcription factors in plant responses \nto pathogens. BMC Plant Biol. 2008; 8:68. https://doi.org/10.1186/1471-2229-8-68.\nLei, R., et al. Arabidopsis WRKY2 and WRKY34 transcription factors interact with VQ20 protein to \nmodulate pollen development and function. Plant J. 2017; 91(6):962-76.\nLi, C., et al. Molecular cloning and expression analysis of WRKY transcription factor genes in \nSalvia miltiorrhiza. BMC Genomics. 2015; 16: 200. https://doi.org/10.1186/s12864-015-\n1411-x.\nLing, J., et al. Genome-wide analysis of WRKY gene family in Cucumis sativus. BMC Genomics. \n2011; 12: 471. doi: 10.1186/1471-2164-12-471.\nLobo, M.G., Paull, R.E. Handbook of pineapple technology: postharvest science, processing and \nnutrition. 2017.\nLoukou, A.L., et al. Effect of harvest time on seed oil and protein contents and compositions in the \noleaginous gourd Lagenaria siceraria (Molina) Standl. J. Sci. Food Agric. 2011; 91(11): \n2073–2080.\nLuan, Q., et al. CsWRKY50 mediates defense responses to Pseudoperonospora cubensis infection \nin Cucumis sativus. Plant Science. 2019; 279:59–69. https://doi.org/10. \n1016/j.plantsci.2018.11.002 PMID: 30709494\nLuo, M., et al. MINISEED3 (MINI3), a WRKY family gene, and HAIKU2 (IKU2), a leucine-rich \nrepeat (LRR) KINASE gene, are regulators of seed size in Arabidopsis. Proceedings of the \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\nNational Academy of Science in the United States of America. 2005; 102:17531–17536.\nMashilo, J., et al. Phenotypic and genotypic characterization of bottle gourd [Lagenaria siceraria \n(Molina) Standl.] and implications for breeding:  a Review. Sci. Hortic. 2017; 222, :136– \n144.\nNoutoshi, Y., et al. A single amino acid insertion in the WRKY domain of the Arabidopsis TIR-\nNBS-LRR-WRKY-type disease resistance protein SLH1 (sensitive to low humidity 1) causes \nactivation of defense responses and hypersensitive cell death. Plant J. 2005; 43(6):873–88.\nRaineri, J., et al. The rice transcription factor OsWRKY47 is a positive regulator of the response to \nwater deficit stress. Plant Mol Biol. 2015; 10.1007/s11103-015-0329-7.\nRinerson, C.I., et al. The evolution of WRKY transcription factors. BMC Plant Biol. 2015; 15:66. \nSafder, I., et al. Identification and analysis of the structure, expression and nucleotide polymorphism \nof the GPAT gene family in rice. Plant Gene. 2021; 26:100290.\nShah, B.N., et al. Phytopharmacological profile of Lagenaria siceraria: a review. Asian J. Plant \nSci. 2010; 9(3): 152–157.\nShen, Q. H., et al. Nuclear activity of MLA immune receptors links isolate-specific and basal \ndisease-resistance responses. science. 2007; 315(5815):1098-1103.\nSong, H., et al. Genome-wide identification and characterization of WRKY gene family in Peanut. \nFront Plant Sci. 2016; 7:534.\nSong, H., et al. Global analysis of WRKY genes and their response to dehydration and salt stress in \nsoybean. Front Plant Sci. 2016; 7:9.\nWang, M., Vannozzi, A., Wang, G., Liang, Y., et al. Genome and transcriptome analysis of the \ngrapevine (Vitis vinifera L.) WRKY gene family. Horticulture Research. 2014; 1:16.\nWang, P.J., et al. Genome-wide identification of WRKY family genes and their response to abiotic \nstresses in tea plant (Camellia sinensis). Genes & Genomics. 2019; 41(1):17-33\nWang, Y.L., et al. Genome-Wide Analysis of the Rad21/ REC8 Gene Family in Cotton (Gossypium \nspp.). Genes. 2023; 14(5):993.\nWaqas, M., et al. Genome-wide identification and expression analyses of WRKY transcription \nfactor family members from chickpea (Cicer arietinum L.) reveal their role in abiotic stress-\nresponses. Genes Genomics. 2019; 41(4):467–81.\nWu, J., et al. Genome-wide investigation of WRKY transcription factors involved in terminal \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\ndrought stress response in common bean. Front Plant Sci. 2017; 8:380.\nWu, K.L., et al. The WRKY family of transcription factors in rice and Arabidopsis and their origins. \nDNA Research. 2005; 12:9-26.\nWu, S., et al. The bottle gourd genome provides insights into Cucurbitaceae evolution and facilitates \nmapping of a Papaya ring-spot virus resistance locus. Plant J. 2017; 92, :963–975.\nWu, X.L., et al. Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing \nOsWRKY11 under the control of HSP101 promoter. Plant Cell and Reports. 2009; 28:21–30.\nXie, Z., et al. Salicylic acid inhibits gibberellininduced alpha-amylase expression and seed \ngermination via a pathway involving an abscisic-acid-inducible WRKY gene. Plant Mol Biol. \n2007; 64: 293–303.\nYang, X., et al. Cold-responsive miRNAs and their target genes in the wild eggplant species \nSolanum aculeatissimum. BMC Genomics. 2017b; 18:1000.\nYang, Y., et al. Characterization of soybean WRKY gene family and identification of soybean \nWRKY genes that promote resistance to soybean cyst nematode. Scientific Reports. 2017a; \n7:17804.\nYang, Y., et al. Functional analysis of structurally related soybean GmWRKY58 and GmWRKY76 \nin plant growth and development. Journal of Experimental Botany. 2017a; 67:4727–4742.\nYu, D.Q., et al. Evidence for an important role of WRKY DNA binding proteins in the regulation \nof NPR1 gene expression. Plant Cell. 2001; 13:1527–1539.\nYue, H., et al. Transcriptome-wide identification and expression profiles of the WRKY \ntranscription factor family in Broomcorn millet (Panicum miliaceum L.). BMC Genomics. \n2016; 17:343.\nZhu, D., et al. VvWRKY30, a grape WRKY transcription factor, plays a positive regulatory role \nunder salinity stress. Plant Sci. 2019; 280:132–42.\nZou, X., et al. Interactions of two transcriptional repressors and two transcriptional activators in \nmodulating gibberellin signaling in aleurone cells. Plant Physiol. 2008; 148: 176–186.\nZou, Z., et al. Gene structures, evolution and transcriptional profiling of the WRKY gene family in \nCastor Bean (Ricinus communis L.). PLoS One. 2016; 11(2): e0148243.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 2, 2026. ; https://doi.org/10.64898/2025.12.30.696992doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}