Genome-wide identification and expression analysis of the SPL transcription factor family and its response to abiotic stress in Elymus sibiricus

Genome-wide identification and expression analysis of the SPL transcription factor family and its response to abiotic stress in Elymus sibiricus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide identification and expression analysis of the SPL transcription factor family and its response to abiotic stress in Elymus sibiricus Xiang Meng, Jun Tang, Zeliang Ju, Lin Ma, Dengxia Yi, Wen Li, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6304174/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Oct, 2025 Read the published version in BMC Plant Biology → Version 1 posted 27 You are reading this latest preprint version Abstract Background: Elymus sibiricus is widely utilized for establishing of high-yield artificial grasslands due to its remarkale productivity and strong resistance to environmental stresses, making it an excellent forage species. SPL transcription factors play a pivotal role in regulating plant growth, development, and responses to abiotic stress. Although the SPL gene family has been identified in many plant species, its presence and function in Elymus sibiricus remain largely unexplored. Result: This study presents a comprehensive genome-wide identification and analysis of the SPL gene family in E. sibiricus . A total of 37 EsSPL genes were successfully identified. Their chromosomal distribution, gene structure, conserved motifs, cis-acting regulatory elements, and evolutionary relationships were analyzed. Protein-protein interaction network analysis predicted that SOC1 and TOE2 are the primary interacting proteins. Most EsSPL genes exhibited high expression levels in seedling tissues. Additionally, analysis of abiotic stress responses revealed that the expression of multiple EsSPL genes were altered under salt, drought, ABA, and GA treatments. Conclusion: Through sequence homology analyses, 37 SPL genes were identified in E. sibiricus . Most SPL family members exhibited high expression levels in seedling tissues, with EsSPL2 specifically upregulated under four distinct abiotic stress conditions. These findings provide a foundation for understanding the genetic evolution and biological functions of the SPL gene family in E. sibiricus , offering valuable insights for future research and breeding efforts. Elymus sibiricus SPL gene family evolutionary relationships gene expression abiotic stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The SPL (SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE) gene family is a group of plant-specific transcription factors widely distributed among green plants [ 1 ]. SPL genes encode transcription factors characterized by a conserved SBP-box domain, which facilitated DNA binding[ 2 , 3 ]. The number and functional diversity of SPL family members vary across plant species [ 4 ]. For instance, Arabidopsis thaliana prossessed 17 SPL genes [ 5 ]. Dispite differences in their primary distinct protein primary structures, all SPL proteins share a highly conserved SBP domain, approximately 80-amino-acid in length[ 6 ]. SPL transcription factors play crucial roles in diverse plant growth and developmental processes including photoperiod and [ 7 , 8 ], gibberellin signalling pathways [ 9 ], regualtion of meristem-specific genes [ 10 , 11 ], and floral organ genes [ 12 , 13 ], responses to heat and drought stress[ 14 – 16 ], leaf morphogenesis [ 17 , 18 ], and the development of lateral, primary roots and adventitious roots [ 19 ]. Current research on SPL genes function predominantly focuses on model plants such as Arabidopsis thaliana and Oryza sativa . In Arabidopsis , miR156 and SPL genes play opposing roles in regulating flowering time: miR156 overexpression delays flowering, whereas SPL genes promote it. Specifically, AtSPL3 , AtSPL4 , and AtSPL5 play critical roles in both flowering time and nutritional phase transition process. Increased expression of AtSPL3 and AtSPL9 accelerates flowering, while miR156 overexpression suppresses SPL activity, leading to dealyed flowering [ 20 , 21 ]. Furthermore, overexpressing AtSPL10 promoters flowering, whereas spl1/spl2 / spl3 triple mutants exhibit delayed flowering compared to the wild type. Further studies indicate that AtSPL10 regulates downstream targets such as MED25, which in turn influences flowering-related genes like FUL and LFY [ 22 ]. Beyond flowing regulation, SPL genes also contribute to stress tolerance and plant architecture. SPL1 and SPL12 are essential for floral organ tolerance to high-temperature stress in Arabidopsis . Transgenic plants overexprssion these genes show enhanced resistance to both extreme and moderate heat stress. In rice, OsSPL3 regulates crown root development [ 23 ]. While OsSPL13 (GLW7) positively affects glume cell size, thereby enhancing grain length and yield [ 24 ]. OsSPL14 ( IPA1 ) (Ideal Plant Architecture 1), a key regulator of plant architecture, not only improves yield but also enhances disease resistance in rice [ 25 , 26 ]. SPL genes regulate various aspects of plant architecture, including inflorescences, pedicel length, floral organ size and early flower development. In Antirrhinum majus (snapdragon), SBP1 and SBP2 interact with the promoter of SQUA , regulating early flower development by influencing SQUA expression [ 27 ]. Moreover, in East China grape, VpSBP16 enhances salt and drought tolerance by modulating SOS and ROS signaling pathways when overexpressed in Arabidopsis . This improvement is observed across mutiple growth stages, including seed germination, seedling growth, and mature plants [ 28 ]. In Malus Xiaojinensis (small crabapple), MdSPL26 interacts with MxTIFY9 , which is induced by exogenous hormone IBA, to jointly suppress the MxHB13 expression. This interaction regulates MxABCB19-2 by binding to its promoter, forming a regulatory pathway that enhances cutting rooting efficiency and promotes primary root growth [ 29 ]. Elymus sibiricus , a heterologous tetraploid plant, is widely distributed across the Eurasian continent, exhibiting remarkable genetic diversity and strong ecological adaptability [ 30 ]. It is particularly valuable for establishing high-yield artificial grasslands, especially in the Tibetan Plateau and northern China. Additionally, E. sibiricus plays a crucial role in restoring and improving natural grasslands, contrebuting to enhanced ecological conditions and increased grassland productivity [ 31 ]. Its high yield and resilience to environmental stressors make it an exceptional forage species, supporting the development of grassland-based livestock farming and ecological restoration efforts. Recently, the reference genome sequence of E. Sibiricus was published, providing valuable insights into its genetic basis and evolutionary history through in-depth genome and population genomic analyses [ 32 , 33 ]. This high-quality genomic reserach establishes a solid scientific foundation for future breeding programs and practical applications of E. Sibiricus. In this study, the SPL (SQUAMOSA promoter-binding protein-like) genes were systematically exploration, identified, and analyzed in E. sibiricus by using its genome as a reference. A total of 37 EsSPL genes were identified, and their evolutionary relationships were examined in comparison with SPL genes from various species, including Arabidopsis thaliana , Solanum lycopersicum , Oryza sativa , Sorghum bicolo , Zea mays , Brachypodium distachyon and Triticum aestivum . Phylogenenic analysis revealed that E. sibiricus shares a closer evolutionary relationship with wheat.Additionally, chromosomal distribution, gene duplication events, cis-acting regulatory elements, gene structures, and conserved motifs of EsSPL family were investigated. Notably, 16 EsSPL genes were found to contain recognition sites for miR156. suggesting thier potential regulation bu this microRNA. Analysis of cis-acting regulatory elements in the promoter regions revealed an enrichment of hormone response elements, indicating their possible roles in hormone signaling pathways. Furthermore, gene expression analysis demonstrated that most EsSPL genes exhibit high expression levels in seedling tissues. Specifically, EsSPL2 was signicantly upregulated under four different types of abiotic stress, suggesting its involvement in stress rsponses. In summary, this comprehensive analysis provided valuable insights into the SPL gene family in Elymus sibiricus , highlighting their crucial roles in growth ,development and stress adaptation. Our findings not only establish a foundation for future functional studies of EsSPL genes but also but also contribute to a deeper understanding of the genetic characteristics and potential applications of E. sibiricus . 2. Material and methods 2.1 Identification and chromosomal localization of Elymus sibiricus SPL genes In this study, the genomic data for E. sibiricus was provided by the Scientific Data ( https://www.nature.com/articles/s41597-024-03622-4 provided on September 6th, 2024)[ 33 ]. The Arabidopsis AtSPL sequences were retrieved from the TAIR database ( http://www.arabidopsis.org/ ). To identifiy EsSPL genes, BLASTp analysis was performed in TBtools using AtSPL sequences as queries. Additionally, conserved domain analysisi was conducted using the CDD-Search database ( https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi ), while the InterPro database ( https://www.ebi.ac.uk/interpro/ ) was utilized to screen for SBP-domain and non-SBP-domain proteins. Pfam model files were downloaded from Pfam database ( https://www.ebi.ac.uk/interpro/download/Pfam/ ). using these resources, TBtools confirmed that the E.sibiricus SPL( EsSPL ) gene family contains SBP domains (PF03110). The chromosomal localization of EsSPL genes was determained based on genome annotation data for E. sibiricus. Additionally, The physicochemical properties of EsSPL proteins were analyzed using the ExPASy protParam tool ( http://web.expasy.org/ ). This analysis included parameters such as amino acid length, molecular weight (MW), isoelectric point (PI), instability index, aliphatic index, and average hydrophobicity (GRAVY). 2.2 Phylogenetic analysis of the EsSPL gene family SPL amino acids sequences were obtained from multiple databases for Arabidopsis thaliana , Solanum lycopersicum , Oryza sativa , Sorghum bicolor , Brachypodium distachyon , Triticum: aestivum , and Zea mays . These sequences were obtained from TAIR (The Arabidopsis Information Resource), the Rice Genome Annotation Project, the Sol Genomics Network, and GIGADB (Genomic Data Commons for Agriculture http://www.gigadb.org .) To investigate the evolutionary relationships among the EsSPL gene family members, a comprehensive phylogenetic tree was constructed. For further functional analysis, additional SPL amino acid sequences from the same species were retrieves from NCBI (National Center for Biotechnology Information https://www.ncbi.nlm.nih.gov/ ), Pear MODB (a molecular database for plant sequences http://www.plantgdb.org/ ), Plant TFDB (a comprehensive database for plant transcription factors http://planttfdb.gao-lab.org/ ), and TAIR. The phylogenetic tree was generated using the Neighbor-Joining (NJ) method in MEGA 11.0 software, with 1,000 bootstrap replications to ensure statistical robustness. The resulting tree was then visualized using the iTOL online platform ( http://itol.embl.de/ ) to provide a clear and structured representation. 2.3 Gene structure and multiple sequence alignment analysis Motif analysis of the EsSPL gene family proteins was conducted using the MEME tool ( http://meme-suite.org/tools/meme ) as described in [ 18 ], A totle of 37 motifs were identified. Additionally, the gene structures and conserved domains of EsSPL family members were analyzed using TBtools to gain a comprehensive understanding of their characteristics. 2.4 Gene duplication and synteny analysis of the EsSPL gene family The chromosomal location and mapping information for the EsSPL gene family members were obtained from the E. sibiricus genome annotation file (GFF3 format) using TBtools. This enabled the visualization of gene distribution across chromosomes, as described in Chen study[ 34 ]. To explore syntenic relationships, genonmic sequences of A. thaliana and Triticum aestivum were downloaded from the Phytozome database ( https://phytozome-next.jgi.doe.gov/ ). Synteny analysis was then performed using the One Step MCScanX function in TBtools, comparing E. sibiricus with Arabidopsis and wheat. The analysis parameters were set to a 'Num of Blast Hits' of 4 and an E-value threshold of 1e-10 to ensure accuracy and reliability. 2.5 Identification of cis-acting regulatory elements in the promoters and prediction of protein-protein interactions Below is a paragraph from an academic paper. Polish the writing to meet the academic style,improve the spelling, grammar, clarity, concision and overall readability. When necessary, rewrite the whole sentence. List the modified paragraphs. Furthermore, list all modification and explain the reasons to do so in markdown table. Paragraph : To investigate the potential functions and expression regulation mechanisms of the EsSPL genes, the 2000 bp upstream promoter sequences of these genes were extracted and uploaded to NCBI. The PlantCARE database (accessed on September 14, 2024, http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) and TBtools were used for visualization [ 35 ]. Protein-protein interaction predictions were carried out using the online STRING database (accessed on September 14, 2024, https://cn.string-db.org/ ). Interaction protein information was retrieved from UniProt ( https://www.uniprot.org/ , accessed on September 14, 2024). 2.6 Growth conditions and stress treatments of Elymus sibiricus The materials of Elymus sibiricus utilized in this experiment were provided by the College of Animal Science and Veterinary Medicine at Qinghai University, China. These plant materials were cultivated within an artificial climate chamber at the Institute of Animal Science and Veterinary Medicine of the Chinese Academy of Agricultural Sciences in Beijing, China. Hydroponic seedlings of Elymus sibiricus were grown in a controlled environment with a photoperiod of 16 hours light (25°C) followed by 8 hours darkness (18°C), a light intensity of 250 µmol photons·m-2·s-1, and a relative humidity of 70%, with regular maintenance. For abiotic stress treatments, seedlings two weeks old were utilized. To investigate gene expression levels in various tissues, roots, stems, leaves, and spikes were gathered at the heading stage for expression analysis. Additionally, seeds and young roots and seedlings of Elymus sibiricus , two weeks old, were sampled for expression analysis as well. All samples were subjected to three biological replicates. The data was visualized using Amazing Heat Map software to generate heatmaps that offer a clear representation of the experimental results. Regarding abiotic stress treatments, two-week-old Elymus sibiricus seedlings were exposed to salt (200 mM NaCl), drought (20% PEG6000), abscisic acid (ABA at 0.1 mM), and gibberellin (GA at 0.1 mM). Samples were collected at various time points: 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 100 h. All experimental procedures were conducted with three biological replicates to ensure accuracy and reliability. 2.7 Total RNA extraction and qPCR analysis Total RNA was extracted utilizing the Trizol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer's prescribed protocol. Subsequently, genomic DNA contamination was eliminated through DNase digestion employing the services of Promega (Madison, WI, USA). The synthesis of the first-strand cDNA was accomplished using M-MLV reverse transcriptase (also provided by Promega) with 1 µg of the total RNA as a template. RT-qPCR analysis was performed on the CFX96 Touch™ Real-Time PCR Detection System from Bio-Rad (Hercules, CA, USA). The PCR amplification parameters were set as: an initial denaturation at 95°C for 2 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 40 seconds. A final cycle consisted of denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 15 seconds. The alfalfa actin gene was utilized as the internal reference, and relative gene expression was analyzed using the 2^-∆∆Ct methodology. All experimental procedures were repeated with three biological replicates and three technical replicates to ensure reliability. Primer design was done using Primer 5.0 software for accurate and consistent results. 2.8 Data analysis The statistical analysis was conducted utilizing the one-way analysis of variance (ANOVA) within the SPSS software version 23.0. The data was presented in the form of mean values accompanied by their corresponding standard deviations. Graphs were created and thoroughly analyzed using GraphPad Prism 10.1.1 software. Any observed differences were deemed statistically significant when the P-value was below 0.05. 3. Results 3.1 Genome-wide identification of the SPL gene family in Elymus sibiricus Using comparative genomics techniques, a total of 37 candidate genes were identified in the E. sibiricus genome [ 33 ]. These genes were named based on their sequence homology with corresponding Arabidopsis SPL proteins. The key characteristics of the EsSPL gene family, including TIGR loci, chromosomal locations, coding and amino acid sequence lengths, molecular weights, theoretical isoelectric points, instability indices, aliphatic indices, and the hydrophilicity, are summarized in Table 1 . The amino acid lengths of EsSPL proteins exhibit significant variation, ranging from 192 to 1,129 residues. Their molecular weight span from 20,156.51 to 123,837.02. The theoretical isoelectric points value range from 5.49 to 9.99, while instability indices vary between 51.57 and 72.05, indicating that these proteins maybe unstable. Furthermore, the aliphatic index varies range from 41.47 to 80.84, reflecting differences in thermostability. Importantly, the average grand hydropaphy(GRAVY) scores of EsSPL proteins are negative, suggesting that they are predominantly hydrophilic. Table 1 List and detailed information of identified SPL genes in Elymus sibiricus . Gene Name Gene ID Chr CDS Length Pep Length Molecular Weight(kDa) Theoretical pI Instability Index Aliphatic Index Grand Average of Hydropathicity EsSPL1 evm.model.Chr01.9142 1 579 192 20156.51 9.99 57.75 59.69 -0.627 EsSPL2 evm.model.Chr01.18876 1 966 321 33678.91 9.36 53.2 58.35 -0.459 EsSPL3 evm.model.Chr01.21724 1 1230 409 44107.57 7.79 57.36 49.02 -0.779 EsSPL4 evm.model.Chr02.9866 2 579 192 20175.47 9.87 62.39 58.65 -0.641 EsSPL5 evm.model.Chr02.18548 2 1386 461 47970.38 9.73 53.8 67.18 -0.254 EsSPL6 evm.model.Chr02.21396 2 1224 407 43930.34 7.05 57.01 48.8 -0.779 EsSPL7 evm.model.Chr03.12983 3 2580 859 94207.19 5.79 53.9 79.7 -0.31 EsSPL8 evm.model.Chr04.13338 4 2580 859 94063.92 5.7 51.57 80.84 -0.28 EsSPL9 evm.model.Chr05.4949 5 1557 518 55294.21 9.32 61.64 60.75 -0.454 EsSPL10 evm.model.Chr06.5732 6 1209 402 42334.95 9.04 58.85 54.75 -0.538 EsSPL11 evm.model.Chr07.7740 7 2514 837 92845.35 9.01 62.24 77.41 -0.388 EsSPL12 evm.model.Chr07.9472 7 1257 418 43461.13 8.85 55.22 50.98 -0.615 EsSPL13 evm.model.Chr07.9649 7 3390 1129 123837.02 7.06 54.04 72.93 -0.536 EsSPL14 evm.model.Chr07.9826 7 1095 364 38309.45 6.7 63.54 57.47 -0.493 EsSPL15 evm.model.Chr07.21378 7 1215 404 43488.89 9.16 60.22 50.89 -0.562 EsSPL16 evm.model.Chr07.21380 7 1257 418 44408.6 9.05 56 50.38 -0.609 EsSPL17 evm.model.Chr08.7950 8 2523 840 93125.69 8.54 59.21 80.62 -0.353 EsSPL18 evm.model.Chr08.9588 8 1146 381 39992.05 9.02 54.88 46.93 -0.755 EsSPL19 evm.model.Chr08.9908 8 3390 1129 123643.82 6.85 52.66 74.13 -0.511 EsSPL20 evm.model.Chr08.10315 8 1275 424 45158.16 6.75 60.14 57.88 -0.492 EsSPL21 evm.model.Chr08.20197 8 1197 398 43199.01 9.59 52.26 60.7 -0.487 EsSPL22 evm.model.Chr08.20224 8 1185 394 42515.77 8.9 60.12 52.44 -0.588 EsSPL23 evm.model.Chr08.20226 8 1251 416 44490.76 9.04 55.53 52.74 -0.604 EsSPL24 evm.model.Chr09.13491 9 1227 408 42487.41 8.39 61.73 41.47 -0.739 EsSPL25 evm.model.Chr09.14379 9 1290 429 44773.98 6.88 63.82 57.46 -0.378 EsSPL26 evm.model.Chr09.21556 9 2892 963 105347.61 5.57 54.13 78.71 -0.311 EsSPL27 evm.model.Chr10.13639 10 1203 400 41905.95 8.74 60.75 42.55 -0.705 EsSPL28 evm.model.Chr10.14363 10 1302 433 45246.59 6.88 61.07 55.8 -0.371 EsSPL29 evm.model.Chr10.21624 10 2898 965 105583.95 5.49 53.11 78.85 -0.301 EsSPL30 evm.model.Chr11.2280 11 1227 408 43789.34 8.71 60.82 60.59 -0.388 EsSPL31 evm.model.Chr11.5748 11 1422 473 51073.13 9.1 57.96 52.45 -0.619 EsSPL32 evm.model.Chr11.7263 11 1350 449 48342.73 7 72.05 47.95 -0.631 EsSPL33 evm.model.Chr11.7419 11 981 326 35316.67 9.34 67.48 53.9 -0.668 EsSPL34 evm.model.Chr12.4808 12 1422 473 51077.04 9.18 62.32 50.78 -0.645 EsSPL35 evm.model.Chr12.6316 12 1461 486 52010.72 8.59 63.11 63.15 -0.345 EsSPL36 evm.model.Chr12.6799 12 978 325 34954.24 9.34 61.04 54.4 -0.611 EsSPL37 evm.model.Chr14.18350 14 933 310 33707.14 6.82 61.52 56.94 -0.535 3.2 Phylogenetic tree analysis of the SPL gene family in Elymus sibiricus To explore the evolutionary relationships of SPL proteins in E. sibiricus , a comprehensive phylogenetic analysis was conducted using multiple model plants species, including A. thaliana , S. lycopersicum, O. sativa, S. bicolor, B. distachyon , T. aestivum , and Z. mays . (Fig. 1 ). To further explore the structural complexity of SPL gene family, a Neighbor-Joining (NJ) phylogenetic tree was constructed based on SPL protein sequences from E. sibiricus (19), A. thaliana (17), S. lycopersicum (15), O. sativa (19), S. bicolor (18), Z. mays (31), B. distachyon (18), and T. aestivum (56). The phylogenetic analysis grouped these SPL genes into seven distinct clades, revealing evolutionary patterns among species. Notably, in Group 6, EsSPL genes clustered closely with wheat TaSPL genes, suggesting a strong co-evolutionary relationship between E. sibiricus and T. aestivum . Specifically, the homologous genes such as EsSPL15 , EsSPL16 , EsSPL21 , EsSPL22 , and EsSPL23 exhibited high sequence similarity with T. aestivum TaSPL genes (e.g., TaSPL 16A, TaSPL 16D ), implying potential functional conservation and shared evolutionary trajectories that warrant further investigation. Furthermore, the analysis revealed a significant expanaion of the SPL gene family from lower to higher plants.This expansion is accompanied by extensive gene duplication events, which likely contributed to the evolutionary success of flowering plants by enhancing their adaptability to diverse and fluctuating environmental conditions [ 36 ]. 3.3 Gene structure analysis of the SPL gene family in Elymus sibiricus To further explore the functional diversity of SPL-associated candidate genes in E. sibiricus , we analyzed their conserved motifs, domains, and gene structures (Fig. 2 ). Using the MEME suite for multiple sequence alignments, the sequence markers that target the SBP domain in E. sibiricus were identified. This approach facilitated the comprehensive assessment of the conservation of homologous domain sequences, particularly in relation to the SBP domain. SPL proteins are structurally characterized by two zinc ion-binding motifs: C3H (Cys-Cys-Cys-His) and C2HC (Cys-Cys-His-Cys). Additionally, a Nuclear Localization Signal (NLS) overlaps with the second zinc finger structure at the carboxyl terminus of the SBP domain. Our analysis revealed that Motif 1 and Motif 2 in E. sibiricus domain correspond to Zn finger motifs, both of which are present in all EsSPL proteins, highlighting a high degree of sequence conservation. Notably, EsSPL37 is the only excpeption, as it lacks Motif 2 (Fig. 2 A). To investigate post-transcriptional regulation, we alighed complementary sequences of miR156 with the multiple sequence alignment of EsSPL genes in (Supplementary Fig. 1). The analysis identified miR156 recognition sites in 16 out of 37 EsSPL genes, acounting for 43.2% of the gene family. Interestingly, these recognition sites were consistently located within motif 7, suggesting a conserved regulatory mechaism. Beyond the SBP domain, certain EsSPL proteins exhibited additional structural features. Specially, EsSPL26 and EsSPL29 possess an extra ANKYR domain, which may imply functional diversification (Fig. 2 B). Analysis of the intron-exon structures showed that the the number of introns among EsSPL genes varies from 2 to 11, with a maximum of three exons (Fig. 2 C). Collectively, these findings provide critical insights into the structural conservation and functional specificity of SPL-related candidate genes in E. sibiricus. The presence of conserved domains, motifs, and gene structures suggests their essential roles in various biological processes, highlighting their evolutionary significance and regulatory potential. 3.4 Chromosomal location and synteny analysis Through mapping the open reading frames (ORFs) of all identified EsSPL genes to their respective chromosomes, the distribution of the SPL gene family in Elymus sibiricus were determined (Fig. 3 A). The results indicated a non-uniform distribution of EsSPL genes across the 14 chromosomes of E. sibiricus . Specifically, chromosome 8 contained the highest number of SPL genes, with 7 identified, followed by chromosomes 7 (6 genes), chromosome 11 (4 genes), and chromosome1and 2( (3 genes each). In contrast, chromosome 3, 4, 5, 6, and 14 contained only aa single EsSPL gene, while chromosome 13 lacked any EsSPL genes entirely. Synteny analysis within E. sibiricus identified 19 gene pairs., demonstrating that members of the EsSPL gene family predominantly exist in tandem. This pattern is likely a comsequence of the allopolyploid nature of E. sibiricus , which has led to the retention of duplicated genes (Fig. 3 B). To further elucidate the evolutionary relationships of the EsSPL gene family, a comparative synteny map was constructed among A. thaliana , E. sibiricus , and T. aestivum . Interestingly, Arabidopsis shares 6 orthologous genes pairs with E. sibiricus , whereas E. sibiricus shares a significantly higher number of orthologous genes pairs (25) with wheat (Fig. 3 C). Calculation of the nonsynonymous-tosynonymous substitution rate ratio (Ka/Ks) for paralogous gene pairs revealed that all values were less than 1, Indicated that these genes are under purifying selection and have conserved functions throughout evolution (Table 2 ). Additionally, the estimated divergence times for both orthologous and paralogous gene pairs provide valuable evolutionary insights. The divergence time for orthologous gene pairs ranged from 37.83 to 168.38 million years ago (MYA), whereas t paralogous gene pairs diverged between36.84 and 246.61 MYA. These findings suggest that the expansion of the SPL gene family in E. sibiricus is likely a result of an ancient whole-genome duplication (WGD) event, followed by subsequent segmental duplications (Table 2 ). Table 2 Ka and Ks values of homologous gene pairs in Elymus sibiricus . Ka Ks Ka/Ks Purify selection Duplication type Time = Ks/2λ ( MYAa) EsSPL1/EsSPL2 0.2969 0.9044 0.3283 Yes Segmental 74.74 EsSPL2/EsSPL9 0.3979 0.712 0.5588 Yes Segmental 58.84 EsSPL3/EsSPL15 0.3754 0.8177 0.4591 Yes Segmental 67.58 EsSPL4/EsSPL21 0.4454 0.8534 0.5219 Yes Segmental 70.53 EsSPL12/EsSPL20 0.3821 1.0634 0.3593 Yes Segmental 87.88 EsSPL15/EsSPL25 0.337 0.9228 0.3652 Yes Segmental 76.26 EsSPL15/EsSPL33 0.4147 0.8941 0.4638 Yes Segmental 73.89 EsSPL15/EsSPL36 0.4154 0.6889 0.6030 Yes Segmental 56.93 EsSPL16/EsSPL20 0.3687 2.984 0.1236 Yes Segmental 246.61 EsSPL20/EsSPL30 0.1366 0.5716 0.2390 Yes Segmental 47.24 EsSPL22/EsSPL33 0.4147 0.9751 0.4253 Yes Segmental 80.59 EsSPL23/EsSPL36 0.4277 0.8875 0.4819 Yes Segmental 73.35 EsSPL24/EsSPL30 0.5296 2.4342 0.2176 Yes Segmental 201.17 EsSPL30/EsSPL37 0.2636 0.4578 0.5758 Yes Segmental 37.83 3.5 Protein-protein interaction network analysis of the SPL gene family in E. sibiricus A protein-protein interaction (PPI) network was constructed and analyzed using STRING network modeling technology, revealing key topological features. The analysis identified 37 SPL family proteins predicted to interac with a diverse range of proteins, suggesting their potential s t roles in pathways related to plant hormone signaling, environmental stress responses, as well as growth and developmental processes (Fig. 4 ). Notably, homologous proteins exhibit similar interaction patterns, as observed in EsSPL1 and EsSPL4, EsSPL2 and EsSPL16, and various other EsSPL proteins pairs. This indicates that these protein pairs may share conserved interaction domains, highlighting substantial connectivity within the EsSPL network and its intercations with other proteins. Additionally, SOC1 and TOE2 frequently appear in these interaction networks. Previous studies have demonstrated that SOC1 gene functions as a central integrator of flowering signals, intergrating inputs from multiple pathways -including photoperiod, autonomous, gibberellin, vernalization, and age-dependent pathways- to regulate flowering time. Similarly, cytokinien modifies the transition from vegetative to reproductive growth via miR172 and its target genes TOE1 and TOE2[ 37 , 38 ]. This analysis provides deeper understanding of the functional regulation of interacting proteins within the SPL gene family,offering a comprehensive perspective on their intricate interactions and roles in biological processes. 3.6 Identification of cis-acting regulatory elements in the promoters of the SPL gene family in Elymus sibiricus Using Plant CARE to analyze the promoter regions of EsSPL genes, a total of 966 cis-acting regulatory elements were identified (Fig. 5 B). Among them, 369 cis-acting regulatory elements were associated with hormone response regulation (Fig. 5 C). Four hormone-responsive motifs were detected, including methyl jasmonate response elements (CGTCA-motif), salicylic acid response elements (TCA-element), gibberellin response elements (P-box), and abscisic acid (ABA) response elements (ABRE). Additionally, various stress-responsive elements, were identified such as drought-related regulatory elements (MBS), low-temperature response elements (LTR), anaerobic response elements (ARE), and defense and stress response elements (TC-rich) etc. These cis-acting regulatory elements were classified into four major categories: environmental stress response elements, hormone response elements, development-related elements, and light response elements (Fig. 5 B). Among the environmental stress response elements, anaerobic response elements were the most abundant, accounting for 33.2% of the total. Within the hormone response element, ABRE motif were the most prevalent, constituting 64.8%, followed by CGTCA-motif elements at 27.4%. Among the five identified development-related elements, the CAT-box element was the most frequent(( 40.6%), followed by the O2-site element (29.2%). For light light-responsive elements, the G-box element was the most common(52.8%), followed by the SPL element ( 21.2%) (Fig. 5 B). Notably, the distribution of cis-acting regulatory elements varied among different EsSPL genes. EsSPL20 exhibited a higher proportion of hormone and light response cis-acting regulatory elements, whereas EsSPL3 and EsSPL6 contained a greater number of hormone response elements. In cntrast EsSPL28 harbored more environmental stress response elements (Fig. 5 C). 3.7 Tissue-specific expression of SPL -Related candidate genes in E. sibiricus To investigate the potential functions of SPL -related candidate genes in different tissues of E. sibiricus and determine whether these genes serve broad housekeeping roles or paerticipate in tissue-specific regulatory mechanisms, a tissue-specific expression analysis was performed(Fig. 6 ). The results revealed that EsSPL33 exhibits strong expression in root tissues, while EsSPL15 and EsSPL16 are highly expressed in stem. Similarly, EsSPL4 demonstrates elevated expression in leaves. Several genes, including EsSPL8, EsSPL10, EsSPL25, EsSPL28, EsSPL34 , and EsSPL36 , displayelevated expression levels in spike tissues. Notably, a large subset of EsSPL genes ( EsSPL1, EsSPL2, EsSPL3, EsSPL6, EsSPL7, EsSPL13, EsSPL14, EsSPL17, EsSPL18, EsSPL21, EsSPL24, EsSPL26, EsSPL29, EsSPL31, EsSPL35 , and EsSPL37 ) showed predominant expression in seedlings. Additionally, EsSPL9, EsSPL23, EsSPL27 , and EsSPL35 exhibit strong expression in young roots, whereas EsSPL5 , EsSPL11, EsSPL12, EsSPL19, EsSPL20, EsSPL22, EsSPL30 , and EsSPL32 are highly expressed in seeds. It is worth noting that, similar to Setaria italic (foxtail millet), most SPL gene family members in E. sibiricus exhibit high expression levels in seedlings, suggesting a conserved regulatory role during plant early development stages. 3.8 Expression of SPL -related candidate genes in E. sibiricus in response to salt, drought, ABA, and GA stress To assess the role of SPL -related candidate genes in the stress response of E. sibiricus , their expression levels were analyzed following exposure to abiotic stress conditions. Two-week-old plants were subjected to hydroponic treatments, 200 mM NaCl (salt stress), 20% PEG6000(drought stress), 0.1 mM abscisic acid (ABA )), and 0.1 mM gibberellin (GA).Treatments were administered for up to 100 hours, with samples collected at intervals of 3, 6, 12, 24, 48, 72, and 100 hours. For each treatment and time point, three plants were carefully harvested, and gene expression ws quatified using three biological replicates to ensure accuracy Under salt stress conditions, most SPL genes exhibited a downregulation trend over time. Specifically, EsSPL5 , EsSPL6, EsSPL13, EsSPL16, EsSPL19, EsSPL25, EsSPL26, EsSPL27 , and EsSPL28 were significantly downregulated compared to their pre-treatment expression levels. In contrast, EsSPL2, EsSPL7, EsSPL10, EsSPL11, EsSPL14, EsSPL17, EsSPL20, EsSPL21, EsSPL23, EsSPL33 , and EsSPL34 exhibited an initial increase followed by a decline. Notably, EsSPL4 and EsSPL9 genes showed sustained upregulation, with expression levels increasing 5.45-fold and 6.55-fold, respectively, compared to pre-treatment levels ( Fig. 7 ). These findings suggested that EsSPL4 and EsSPL9 may play significantly roles in negative regulation under salt stress and could serve as potential targets for future studies on salt stress adaptation. Similarly, under drought stress conditions, many SPL genes exhibited an initial upregulation followed by a gradual decrease over time. Specifically, EsSPL2, EsSPL3, EsSPL10, EsSPL11, EsSPL12, EsSPL13, EsSPL14 and EsSPL1 8, were significantly upregulated. Among them, EsSPL8 and EsSPL16 genes demonstrated the most pronounced increases, with expression levels rising 5.66-fold and 4.75-fold, respectively. compared to pre-treatment levels (Fig. 7 ). These results indicated that SPL gene family members play a crucial role in drought stress regulation, particularly EsSPL8 and EsSPL16 , which may contribute to osmotic stress adaptation and root development processes. Under ABA treatment, the expression levels of EsSPL17, EsSPL22 , and EsSPL36 gradually decreased over time, indicating significant downregulation compared to pre-treatment levels. In contrast, EsSPL6, EsSPL11, EsSPL12, EsSPL15, EsSPL27 , EsSPL30 , EsSPL31, EsSPL33 , and EsSPL34 exhibited an initial upregulation followed by a subaequent decline (Fig. 8 ). Specifically, EsSPL2 displayed a substantial regulation of 5.52-fold compared to pre-treatment levels. Interestingly, the upregulation of EsSPL2, EsSPL11, EsSPL12 , and EsSPL27 under ABA treatment was consistent with their expression patterns observed in the drought treatment, suggesting that SPL transcription factors may play a crucial role in ABA-mediated stress responses GA (gibberellin) regulates various plant growth and developmental processes, including seed germination, flower organ development, etc. Under GA treatment, the expression levels of several genes including EsSPL7, EsSPL8, EsSPL9, EsSPL10, EsSPL15, EsSPL16, EsSPL19, EsSPL20, EsSPL22, EsSPL25, EsSPL27, EsSPL32, EsSPL34, EsSPL36 , and EsSPL37 initially increased before gradually declining, indicating a significant upregulation in comparison to their pre-treatment levels. Specifically, EsSPL2, EsSPL14 , and EsSPL17 exhibited the most pronounced increases, with expression levels rising 3.92-fold, and 5.01-fold, and 6.31-fold, respectively (Fig. 8 ). These findings suggested that SPL might be involved in gibberellin signaling transduction[ 39 ], further reinforcing their regulatory roles in plant growth and stress adaptation . 4. Discussion In this study, a total of 37 SPL -related candidate genes were identified in the genome of E. sibiricus andcategorized into eight distinct groups. Comparative analysis with other plant species reveals a diverse gene distribution, including 17 SPL genes from Arabidopsis [ 40 ], 15 from tomato [ 41 ], 17 from sorghum [ 42 ], 19 from rice [ 43 ], and a notably larger count of 31 from maize [ 44 ]. Evolutionary analysis, gene structure examination, and sequence alignment demonstrated classification patterns in E. sibiricus ,similar to those observed in other species. All EsSPL genes contained the SBP domain, while EsSPL26 and EsSPL29 also possessed an Ankyrin domain, consistent with previous findings on the SPL gene family structure in lychee [ 16 ]. Gene duplication is a key driver of genetic evolution [ 45 ]. In E. sibiricus , an evolutionary pattern similar to that of other plants was observed. While most EsSPL genes exist as single-copy genes, several multi-copy genes were identified, including EsSPL1, EsSPL4, EsSPL7 , and EsSPL8 ,as well as EsSPL12, EsSPL16, EsSPL20, EsSPL24 , and EsSPL30 , which are also found across across various species. Notably, duplicated SPL genes in E. sibiricus are distributed across different chromosomes (Fig. 3 ), reflecting their complex evolutionary history. Most SPL regulatory genes appear to have originated from whole-genome duplication (WGD) events, while genes such as EsSPL4, EsSPL8, EsSPL16, EsSPL20 , and EsSPL30 likely arose through segmental duplications (Fig. 3 ). These findings suggested that the expansion of SPL genes is predominantly driven by WGD and segmental duplication. Moreover, interspecies collinearity analysis revealed that 67.57% of Es SPL genes have orthologs in wheat; whereas only 16.22% share orthology with Arabidopsis , highlighting the increasing functional divergence of SPL genes between monocots and dicots. To further explore the evolutionary dynamics of EsSPL genes,, synonymous (Ks) and and nonsynonymous (Ka) substitution rates were calculated for paralogous gene pairs. The Ka/Ks ratio, an indicator of selective pressure [ 46 ] was examined: Ka/Ks ratio> 1 indicates positive selection, Ka/Ks = 1 suggests neutral selection, Ka/Ks< 1 indicates purifying selection. The results showed an average Ka/Ks ratio of 0.41 for paralogous EsSPL gene pairs in E. sibiricus , indicating that the EsSPL genes have undergone purifying selection and have been subject to strong evolutionary constrains. In plants, the exon/intron structure of the SPL gene exhibits low conservation withexon numbers varying significantly. In E. sibiricus, EsSPL genes contain 2 to 11 exons(Fig. 2 ), a range comparable to that in chickpea (2–10 exons) [ 47 , 48 ], sunflower (2–11 exons) [ 49 ], and lychee (2–10 exons) [ 16 ]. Interestingly, homologous EsSPL often often differ in exon count, such as EsSPL32 (3 exons) and EsSPL33 (6 exons), suggesting that exon gain or less has contributed to structural variations during evolution. Despite similar motif sequneces and arrangements among EsSPL genes (Fig. 2 ), substantial motif diversity was observed. Motif 3 and Motif 6 are exclusive to the fifth evolutionary group,, while Motif 4 and Motif 5 are unique to the sixth group. Additionally, Motif 8 is found only in EsSPL14, EsSPL20, EsSPL25, EsSPL28, EsSPL30 , and EsSPL37 . Conversely, the highly conserved Motifs 1 and 2, present in the core regions of EsSPL proteins, likely play a crucial role in their function as transcription factors [ 50 ]. Consequently, the structural diversity of EsSPL proteins is closely linked to their functional divergence, with additional motifs contributing to their specialized roles in E. sibiricus . This study analyzes the promoters of SPL- related candidate gene, identifying 19 cis-acting regulatory elements associated with environmental stress, plant growth, development, hormone response, and light response (Fig. 5 ). Universally prevalent elements include the hypoxia-specific response element (ABRE), linked to meristematic cell division and proliferation (CAT box), light-responsive elements (MRE, GT1-motif) (Hernandez-Garcia and Finer, 2014). Specifically, ABRE and CAT- box elements correlate with meristematic tissue development and hypoxic stress responses [ 5 ].Research by Weits et al. [ 51 ] and Shukla et al. [ 52 ] highlighted the significance of apical meristem development under low oxygen conditions, essential for initiating new leaf formation. Moreover, hypoxia affects lateral root primordia by inhibiting the degradation of key regulatory proteins. Under such conditions, light acts as a signaling factor, activating stem cells through CK signaling and metabolic pathways [ 53 ]. The abundance of developmental elements(ABRE ,CAT-box) in SPL promoters suggests their involvement in plant hormones signalling [ 54 , 55 ], particularly in response to GA, which influences plant height and morphology. Furthermore, key cis-acting regulatory elements (GT-1-motif, CAT –box,GATAT-motif) have been identified in E. sibiricus SPL gene promoters[ 56 ]. SPL transcription factors regulate flowering in in a tandem manner Elymus sibiricus and serve as a target of miR156. Overexpression of OsmiR156b and OsmiR156h in previous studies resulted in dwarfism, reduced inflorescence size, and delayed flowering, emphasizing the pivotal role of miR156-SPL regulation in embryonic development [ 57 ]. Given the importance of SPL expression in flowering, future studies should focus on the functional validation of these cis-acting regulatory elements, exploring SPL interactions touncover novel insights into plant growth, morphology, and developmental processes. In organisms, gene expression precedes and is essential for gene function, with expression patterns intricately linked to gene functions [ 57 ]. The SPL gene family is widely involved in plant growth and development, playing a key role in regulating traits from seedlings to maturity [ 58 , 59 ]. In Arabidopsis , AtSPL10 eliminates de novo shoot regeneration capacity by derepressing the attenuation of cytokinin response in the primary root [ 19 ]. In Rhododendron molle , RmSPL1/7/8/12/13 exhibit significantly divergent expression, suggesting their essential roles in regulating floral bud differentiation [ 60 ]. TaSPL6B recruits TaD53 and TaSPL3 to integrate light and strigolactone signaling pathways, thereby suppressing the key tillering inhibitor TaTB1 and enhancing TaD53 -mediated inhibition of TaSPL3 in T. aestivum [ 61 ]. Similarly, SPL genes also play crucial roles in abiotic stress responses. This study demonstrates that under salt stress, EsSPL4 and EsSPL9 exhibit sustained upregulation and likely function as important negative regulators, suggesting their potential as key targets for future research on salt stress adaptation and genetic enhancement strategies. In studies on salt tolerance in Codonopsis pilosula , CpSPL5 and CpSPL8 were identified as negative regulators of salt stress resistance, indicating that members of the SPL gene family are involved in the plant SOS pathway [ 62 ]. In Arachis hypogaea (peanut) salt tolerance studies, AhSPL5 , AhSPL16 , AhSPL25 , and AhSPL36 were found to be upregulated under both drought and salinity conditions [ 63 ]. In this study, EsSPL8 and EsSPL16 showed the most significant unregulation under drought stress. However, no consistent expression patterns were observed between salt and drought treatments, which might be attributed to differences in stress application conditions. In Mangifera indica (mango) stress resistance studies, overexpression of MiSPL3a and MiSPL3b in transgenic Arabidopsis conferred enhanced drought tolerance and ABA-induced stress resistance [ 64 ]. Under ABA treatment, EsSPL2 , EsSPL11 , EsSPL12 , and EsSPL27 displayed expression patterns consistent with those observed under drought stress, suggesting that SPL transcription factors play pivotal roles in both drought and ABA-mediated stress responses. Under GA treatment, EsSPL2 , EsSPL14 , and EsSPL17 exhibited the most pronounced upregulation, indicating a potential role in gibberellin signal transduction and further emphasizing their regulatory functions in plant growth and stress adaptation [ 65 ]. Both GA and SPL genes regulate the key flowering integrators such as LFY and SOC1. GA promotes flowering by alleviating DELLA-mediated repression of these genes, while SPL transcription factors directly activate their transcription. This synergistic regulatory mechanism enables plants to fine-tune flowering time in response to internal developmental cues and external environmental signals, thereby ensuring reproductive success. Based on these fondings, we propose that the SPL gene family plays a crucial role in E. sibiricus abiotic stress resistance. Specifically, EsSPL2, EsSPL4, EsSPL8, EsSPL9, EsSPL14, EsSPL16 , and EsSPL17 may function as keymediators of stress tolerance. Notably, EsSPL2 gene consistently exhibited high expression across all stress treatments compared to the control (CK), under the four stress conditions examined, the demonstrates compared to the, indicating its potential as a key regulator in stress resistance mechanisms in E. sibiricus . E. sibiricus may utilize EsSPL2 for direct stress resistance or as a regulatory hub influencing other stress-responsive genes. Determining whether EsSPL2 functions through direct expression or indirect regulatory mechanisms remains a critical avenue for future investigation. 5. conclusion This study represents the first genome-wide investigation of the SPL gene family in E. sibiricus , identifying 37 EsSPL members primarily expanded through segmental duplications. Regulatory analysis revealed the presence of hormone and stress-responsive cis-acting elements in EsSPL promoters, while protein interaction predictions implicated SOC1 and TOE2 as key interacting partners. Expression profiling highlighted tissue-specific expression patterns in seedlings, with EsSPL2 exhibiting both constitutive expression and significant induction under four abiotic stresses. In summary, this research enhances the understanding of the genetic evolution and biological functions of the EsSPL gene family, providing a foundation for future functional characterization and application in stress adaptation studied. Abbreviations SPLSquamosa promoter-binding protein-like IPA1 Ideal Plant Architecture 1 MW Molecular weight PI isoelectric point ABA abscisic acid GA gibberellin Declarations Ethics approval and consent to participate This article does not contain any studies involving human participants or animals performed by the authors. These methods were carried out by relevant guidelines and regulations. Elymus sibiricus used in this study is a prevalent wild species in the Qinghai area. Based on international standards, all the experimental research and field studies on plants, including the collection of plant material were approved by Institute of Animal Sciences. These materials are stored in Institute of Animal Sciences of Chinese Academy of Agricultural Sciences. Consent for publication Not applicable. Availability of data and materials The genome sequences of A. thaliana, S. lycopersicum, O. sativa, S. bicolor, Z. mays, B. distachyon, and T. aestivum were downloaded from Phytozome database (https://phytozome-next.jgi.doe.gov/). The datasets supporting the results of this article are included in the article and Additional files. Competing Interests The authors declare no competing interests. Funding This work was supported by the National Key Research and Development Program of China (2023YFD1200303), the earmarked fund for China Agriculture Research System(CARS-34), the National Crop Germplasm Resources Center (NCGRC-63), and Xinjiang Rural Key Talent Training Project (2022SNGGGCC019). Authors' contributions performed the experiments with the help of D.Y., L.M., X.M.,J.T., X.M., K.X., and W.L. analyzed the sequencing data; X.W., J.T., D.Y., Z.J., W.L., M.H., and W.L. designed the experiments, interpreted the results, and wrote the manuscript. All authors read and approved of the final manuscript. Acknowledgements We thank all our colleagues for providing useful discussions and technical assistance. We are very grateful to the editor and reviewers for critically evaluating the manuscript and providing constructive comments for its improvement. Competing Interests The authors declare that they have no conflict of interest. References Fan E, Liu C, Wang Z, Wang S, Ma W, Lu N, Liu Y, Fu P, Wang R, Lv S, Qu G, Wang J. Genome-wide identification and expression analysis of the SQUAMOSA promoter-binding protein-like (SPL) transcription factor family in Catalpabungei . Int J Mol Sci. 2023;20(1):97. Jiang M, He Y, Chen X, Zhang X, Guo Y, Yang S, Huang J, Traw MB. 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BMC Genomics. 2024;25(1):101. Weits DA, Kunkowska AB, Kamps NCW, Portz KMS, Packbier NK, Nemec Venza Z, Gaillochet C, Lohmann JU, Pedersen O, van Dongen JT, Licausi F. An apical hypoxic niche sets the pace of shoot meristem activity. Nature. 2019;569(7758):714–7. Shukla V, Lombardi L, Iacopino S, Pencik A, Novak O, Perata P, Giuntoli B, Licausi F. Endogenous hypoxia in lateral root primordia controls root architecture by antagonizing auxin signaling in Arabidopsis . Mol Plant. 2019;12(4):538–51. Pfeiffer A, Janocha D, Dong Y, Medzihradszky A, Schöne S, Daum G, Suzaki T, Forner J, Langenecker T, Rempel E, Schmid M, Wirtz M, Hell R, Lohmann JU. Integration of light and metabolic signals for stem cell activation at the shoot apical meristem. Elife. 2016;5:e17023. İlhan E, Büyük İ, İnal B. Transcriptome - scale characterization of salt responsive bean TCP transcription factors. Gene. 2018;5:642:64–73. Rouster J, Leah R, Mundy J, Cameron-Mills V. 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Identification and expression analysis of the SPL gene family during flower bud differentiation in Rhododendron molle. Genes Genomics. 2025;47(2):171–82. Dong F, Song J, Zhang H, Zhang J, Chen Y, Zhou X, Li Y, Ge S, Liu Y. TaSPL6B , a member of the Squamosa promoter binding protein-like family, regulates shoot branching and florescence in Arabidopsis thaliana . BMC Plant Biol. 2024;24(1):708. Li Q, Yang Q, Dong S, Fu F, Xin Y, Kang H, Wu Y, Cao X. Transcription factors CpSPL5 and CpSPL8 negatively regulate salt tolerance in Codonopsis pilosula by inhibiting SOS pathway. Plant J. 2025;121(2):e17205. Sun X, Zhang L, Xu W, Zheng J, Yan M, Zhao M, Wang X, Yin Y. A Comprehensive analysis of the peanut SQUAMOSA promoter binding protein-like gene family and how AhSPL5 enhances salt Tolerance in transgenic Arabidopsis . Plants (Basel). 2024 Apr 93(8):1057. Zhu J, Li Y, Zhang Y, Xia L, Hu W, Huang X, Li K, He X, Luo C. Overexpression of MiSPL3a and MiSPL3b confers early flowering and stress tolerance in Arabidopsis thaliana . Int J Biol Macromol. 2024;262(Pt 1):129913. Yu S, Galvão VC, Zhang YC, Horrer D, Zhang TQ, Hao YH, Feng YQ, Wang S, Schmid M, Wang JW. Gibberellin regulates the Arabidopsis floral transition through miR156-targeted SQUAMOSA promoter binding-like transcription factors. Plant Cell. 2012;24(8):3320–32. Additional Declarations No competing interests reported. Supplementary Files Supplementarytable.xlsx Supplementary Material 1: Supplementary table 1. Classification of promoter cis-elements Supplementary Material 2: Supplementary table 1. Organization of promoter cis-elements Supplementary Material 3: Supplementary table 1. Primer design for SPL gene family SupplementaryFig1.jpg Supplementary Material 4: Supplementary Fig 1. Multiple sequence alignment of the miR156 complementary sequences with the SPL genes of Elymus sibiricus Cite Share Download PDF Status: Published Journal Publication published 07 Oct, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 22 Jul, 2025 Reviewers agreed at journal 21 Jul, 2025 Reviewers agreed at journal 19 Jul, 2025 Reviewers agreed at journal 19 Jul, 2025 Reviewers agreed at journal 18 Jul, 2025 Reviewers agreed at journal 18 Jul, 2025 Reviewers agreed at journal 18 Jul, 2025 Reviewers agreed at journal 17 Jul, 2025 Reviews received at journal 17 Jul, 2025 Reviews received at journal 16 Jul, 2025 Reviews received at journal 16 Jul, 2025 Reviewers agreed at journal 16 Jul, 2025 Reviewers agreed at journal 16 Jul, 2025 Reviewers agreed at journal 16 Jul, 2025 Reviewers agreed at journal 16 Jul, 2025 Reviewers agreed at journal 16 Jul, 2025 Reviewers agreed at journal 10 Jul, 2025 Reviewers agreed at journal 09 Jul, 2025 Reviews received at journal 02 Jul, 2025 Reviewers agreed at journal 18 Jun, 2025 Reviews received at journal 12 May, 2025 Reviewers agreed at journal 12 May, 2025 Reviewers invited by journal 07 May, 2025 Editor invited by journal 06 May, 2025 Editor assigned by journal 15 Apr, 2025 Submission checks completed at journal 14 Apr, 2025 First submitted to journal 14 Apr, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6304174","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":454590625,"identity":"c1f57e8a-673f-498e-8228-aed338dae3ea","order_by":0,"name":"Xiang Meng","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Meng","suffix":""},{"id":454590626,"identity":"9dcbb07d-eead-4c29-ac9e-2cc3f40d87af","order_by":1,"name":"Jun Tang","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Tang","suffix":""},{"id":454590627,"identity":"84c11f15-42dc-4289-a8ef-712841b56b40","order_by":2,"name":"Zeliang Ju","email":"","orcid":"","institution":"Oinghai University","correspondingAuthor":false,"prefix":"","firstName":"Zeliang","middleName":"","lastName":"Ju","suffix":""},{"id":454590628,"identity":"5d6a6341-33f7-4aec-b980-1414acd54cec","order_by":3,"name":"Lin Ma","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Ma","suffix":""},{"id":454590629,"identity":"6c312b8d-1940-4a42-bb3d-2801c12ac2d4","order_by":4,"name":"Dengxia Yi","email":"","orcid":"","institution":"Chinese Academy of Agricultural 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University","correspondingAuthor":false,"prefix":"","firstName":"Miaomiao","middleName":"","lastName":"Huang","suffix":""},{"id":454590635,"identity":"f85469ad-3844-4eb8-9d97-bfe16b883c9c","order_by":8,"name":"Wenhui Liu","email":"","orcid":"","institution":"Oinghai University","correspondingAuthor":false,"prefix":"","firstName":"Wenhui","middleName":"","lastName":"Liu","suffix":""},{"id":454590637,"identity":"40af7be2-fba0-4fa9-9a51-dbd059ec86eb","order_by":9,"name":"Kaiyun Xie","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Kaiyun","middleName":"","lastName":"Xie","suffix":""},{"id":454590638,"identity":"c638befd-3e32-461b-a3f0-2b06bdb031fc","order_by":10,"name":"Xuemin Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDADNgbmAyRrYUsg2R4eA+LUGRw/e/g1b5tNNJ90z8cPP2rqGPhnNxDQciYvzXJmW1pum8zZzZI9xw4zSNw5gF+L2YEcM4OPbYdz2yRytzHwsB1gMJBIIKDl/Bszg8S2/0AtOc8Y//yrI0LLjRzjBx/bDoC0sDHztjET1mJ/440Z44xzyUAtacbSsn2HeSRuENAi2Z9j/JmnzC53/ozkhx/ffKuT459BQAsQsEkg83gIqgcC5g/EqBoFo2AUjIIRDADXakKnpsspmAAAAABJRU5ErkJggg==","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Xuemin","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-03-25 13:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6304174/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6304174/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-07366-0","type":"published","date":"2025-10-07T15:58:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82510956,"identity":"8f2252bf-ae34-42c2-8d77-e2cd4370113a","added_by":"auto","created_at":"2025-05-12 10:40:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10086987,"visible":true,"origin":"","legend":"\u003cp\u003eEvolutionary analysis of the SPL-related candidate genes in \u003cem\u003eElymus sibiricus.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6304174/v1/62c285341ad5790d3e67d1ba.jpg"},{"id":82510085,"identity":"68d4e53e-140d-4706-a6e0-44da970227c8","added_by":"auto","created_at":"2025-05-12 10:32:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2190314,"visible":true,"origin":"","legend":"\u003cp\u003eDetailed information on the conserved motifs, functional domains, and exon-intron organization of \u003cem\u003eSPL\u003c/em\u003e-related candidate genes in \u003cem\u003eElymus sibiricus\u003c/em\u003e where (A) represents conserved motifs, (B) represents functional domains, and (C) represents exon-intron organization.\u003c/p\u003e","description":"","filename":"fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6304174/v1/fe508105bace573578f1874c.jpg"},{"id":82510962,"identity":"7724797e-7fda-439c-83f5-c51c4e655460","added_by":"auto","created_at":"2025-05-12 10:40:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":28401268,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal distribution and synteny analysis of \u003cem\u003eSPL\u003c/em\u003e-related candidate genes in \u003cem\u003eElymus sibiricus\u003c/em\u003e. (A) Spatial distribution of \u003cem\u003eSPL\u003c/em\u003e-related candidate genes on the chromosomes of \u003cem\u003eElymus sibiricus\u003c/em\u003e. (B) Intra-species synteny analysis describing the chromosomal localization of \u003cem\u003eSPL\u003c/em\u003e-related candidate genes. (C) Inter-species synteny analysis illustrating the relationship of SPL-related candidate genes among \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eElymus sibiricus\u003c/em\u003e and wheat. Colored lines represent the syntenic relationships of \u003cem\u003eSPL\u003c/em\u003e-related candidate genes between\u003cem\u003e Elymus sibiricus\u003c/em\u003e and other plant species.\u003c/p\u003e","description":"","filename":"fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6304174/v1/811cf2404a10d660eabec836.jpg"},{"id":82510101,"identity":"e2cd962f-fb2c-484d-9a95-46723c9ef57c","added_by":"auto","created_at":"2025-05-12 10:32:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16451584,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted regulatory network of \u003cem\u003eSPL\u003c/em\u003e and its interacting proteins. Sky blue lines represent data from selected databases; purple lines are experimentally validated interactions; dark blue lines indicate gene co-occurrence; green lines represent text mining results; black lines denote co-expression; light blue lines indicate protein homology.\u003c/p\u003e","description":"","filename":"fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6304174/v1/cb0eff6069cf6b28bcab3f82.jpg"},{"id":82510086,"identity":"1e18499e-138e-4e14-b0b0-2b32b4fee0c2","added_by":"auto","created_at":"2025-05-12 10:32:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8977201,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of the number of cis-acting regulatory elements in \u003cem\u003eSPL\u003c/em\u003e-related candidate genes. Different color shades and corresponding grid numbers represent the number of distinct promoter elements identified in the \u003cem\u003eSPL\u003c/em\u003e-related candidate genes. The multicolored histogram shows the total number of cis-acting regulatory elements in each functional category: blue represents environment stress-related elements, orange represents hormone response elements, yellow represents growth and development-related elements, and green represents light response elements.\u003c/p\u003e","description":"","filename":"fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6304174/v1/d9963d4c51768c787f7387ce.jpg"},{"id":82510959,"identity":"06944e68-1fb3-4ef4-a35b-314fa9e49029","added_by":"auto","created_at":"2025-05-12 10:40:15","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2176709,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of \u003cem\u003eSPL\u003c/em\u003e-related candidate genes in different tissues.\u003c/p\u003e","description":"","filename":"fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6304174/v1/b962a062fe9d40816df4d982.jpg"},{"id":82511419,"identity":"a938d70b-237b-46bc-9d48-1d5d7575d3f9","added_by":"auto","created_at":"2025-05-12 10:48:15","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3966759,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of \u003cem\u003eSPL\u003c/em\u003e-related candidate genes under salt and drought stress treatments. The relative expression levels of these genes were quantified at specific time points: 0, 3, 6, 12, 24, 48, 72, and 100 hours for each treatment. The data presented are means ± standard deviation (SD) calculated from three independent biological replicates. To determine statistical significance, a one-way analysis of variance was conducted, and the results are indicated as follows: \"*\" denotes P \u0026lt; 0.05; \"**\" denotes P \u0026lt; 0.01; and \"***\" denotes P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6304174/v1/4e3d2d7412e83cec895b485e.jpg"},{"id":82511420,"identity":"909686bd-8d40-4993-a09e-02fb98ac25ee","added_by":"auto","created_at":"2025-05-12 10:48:16","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4034667,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of SPL-related candidate genes under ABA and GA treatments. The relative expression levels of these genes were quantified at specific time points: 0, 3, 6, 12, 24, 48, 72, and 100 hours for each treatment. The data presented are means ± standard deviation (SD) calculated from three independent biological replicates. To determine statistical significance, a one-way analysis of variance was conducted, and the results are indicated as follows: \"*\" denotes P \u0026lt; 0.05; \"**\" denotes P \u0026lt; 0.01; and \"***\" denotes P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"fig.8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6304174/v1/933cfdb5dd8f2c0935bac784.jpg"},{"id":93420023,"identity":"f6f2ac56-0ea9-4816-a7d9-54bbdaff0f2d","added_by":"auto","created_at":"2025-10-13 16:09:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":78033438,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6304174/v1/2378334e-9ceb-49fa-b591-9a08b06cf914.pdf"},{"id":82510083,"identity":"0d3850ac-c437-466b-a8ac-706c53450c36","added_by":"auto","created_at":"2025-05-12 10:32:15","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18133,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Material 1: Supplementary table 1.\u003c/strong\u003e Classification of promoter cis-elements\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Material 2: Supplementary table 1.\u003c/strong\u003e Organization of promoter cis-elements\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Material 3: Supplementary table 1.\u003c/strong\u003e Primer design for \u003cem\u003eSPL\u003c/em\u003e gene family\u003c/p\u003e","description":"","filename":"Supplementarytable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6304174/v1/c7ef9419d754297746dcee7c.xlsx"},{"id":82510968,"identity":"6102dbd9-de52-4bf6-ac3d-4e4cec598327","added_by":"auto","created_at":"2025-05-12 10:40:16","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13868079,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Material 4: Supplementary Fig 1. \u003c/strong\u003eMultiple sequence alignment of the miR156 complementary sequences with the \u003cem\u003eSPL\u003c/em\u003e genes of \u003cem\u003eElymus sibiricus\u003c/em\u003e\u003c/p\u003e","description":"","filename":"SupplementaryFig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6304174/v1/17727e8dd12cd2905fed29c2.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide identification and expression analysis of the SPL transcription factor family and its response to abiotic stress in Elymus sibiricus","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe \u003cem\u003eSPL\u003c/em\u003e (SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE) gene family is a group of plant-specific transcription factors widely distributed among green plants [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003eSPL\u003c/em\u003e genes encode transcription factors characterized by a conserved SBP-box domain, which facilitated DNA binding[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The number and functional diversity of SPL family members vary across plant species [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. For instance, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e prossessed 17 SPL genes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Dispite differences in their primary distinct protein primary structures, all SPL proteins share a highly conserved SBP domain, approximately 80-amino-acid in length[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eSPL\u003c/em\u003e transcription factors play crucial roles in diverse plant growth and developmental processes including photoperiod and [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], gibberellin signalling pathways [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], regualtion of meristem-specific genes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and floral organ genes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], responses to heat and drought stress[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], leaf morphogenesis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and the development of lateral, primary roots and adventitious roots [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrent research on \u003cem\u003eSPL\u003c/em\u003e genes function predominantly focuses on model plants such as \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e. In \u003cem\u003eArabidopsis\u003c/em\u003e, miR156 and \u003cem\u003eSPL\u003c/em\u003e genes play opposing roles in regulating flowering time: miR156 overexpression delays flowering, whereas \u003cem\u003eSPL\u003c/em\u003e genes promote it. Specifically, \u003cem\u003eAtSPL3\u003c/em\u003e, \u003cem\u003eAtSPL4\u003c/em\u003e, and \u003cem\u003eAtSPL5\u003c/em\u003e play critical roles in both flowering time and nutritional phase transition process. Increased expression of \u003cem\u003eAtSPL3\u003c/em\u003e and \u003cem\u003eAtSPL9\u003c/em\u003e accelerates flowering, while miR156 overexpression suppresses \u003cem\u003eSPL\u003c/em\u003e activity, leading to dealyed flowering [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, overexpressing \u003cem\u003eAtSPL10\u003c/em\u003e promoters flowering, whereas \u003cem\u003espl1/spl2\u003c/em\u003e/\u003cem\u003espl3\u003c/em\u003e triple mutants exhibit delayed flowering compared to the wild type. Further studies indicate that \u003cem\u003eAtSPL10\u003c/em\u003e regulates downstream targets such as MED25, which in turn influences flowering-related genes like \u003cem\u003eFUL\u003c/em\u003e and \u003cem\u003eLFY\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Beyond flowing regulation, SPL genes also contribute to stress tolerance and plant architecture. \u003cem\u003eSPL1\u003c/em\u003e and \u003cem\u003eSPL12\u003c/em\u003e are essential for floral organ tolerance to high-temperature stress in \u003cem\u003eArabidopsis\u003c/em\u003e. Transgenic plants overexprssion these genes show enhanced resistance to both extreme and moderate heat stress. In rice, \u003cem\u003eOsSPL3\u003c/em\u003e regulates crown root development [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. While \u003cem\u003eOsSPL13\u003c/em\u003e (GLW7) positively affects glume cell size, thereby enhancing grain length and yield [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. \u003cem\u003eOsSPL14\u003c/em\u003e (\u003cem\u003eIPA1\u003c/em\u003e) (Ideal Plant Architecture 1), a key regulator of plant architecture, not only improves yield but also enhances disease resistance in rice [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. \u003cem\u003eSPL\u003c/em\u003e genes regulate various aspects of plant architecture, including inflorescences, pedicel length, floral organ size and early flower development. In \u003cem\u003eAntirrhinum majus\u003c/em\u003e (snapdragon), SBP1 and SBP2 interact with the promoter of \u003cem\u003eSQUA\u003c/em\u003e, regulating early flower development by influencing \u003cem\u003eSQUA\u003c/em\u003e expression [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Moreover, in East China grape, \u003cem\u003eVpSBP16\u003c/em\u003e enhances salt and drought tolerance by modulating SOS and ROS signaling pathways when overexpressed in \u003cem\u003eArabidopsis\u003c/em\u003e. This improvement is observed across mutiple growth stages, including seed germination, seedling growth, and mature plants [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In \u003cem\u003eMalus Xiaojinensis\u003c/em\u003e (small crabapple), \u003cem\u003eMdSPL26\u003c/em\u003e interacts with \u003cem\u003eMxTIFY9\u003c/em\u003e, which is induced by exogenous hormone IBA, to jointly suppress the \u003cem\u003eMxHB13\u003c/em\u003e expression. This interaction regulates \u003cem\u003eMxABCB19-2\u003c/em\u003e by binding to its promoter, forming a regulatory pathway that enhances cutting rooting efficiency and promotes primary root growth [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eElymus sibiricus\u003c/em\u003e, a heterologous tetraploid plant, is widely distributed across the Eurasian continent, exhibiting remarkable genetic diversity and strong ecological adaptability [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It is particularly valuable for establishing high-yield artificial grasslands, especially in the Tibetan Plateau and northern China. Additionally, \u003cem\u003eE. sibiricus\u003c/em\u003e plays a crucial role in restoring and improving natural grasslands, contrebuting to enhanced ecological conditions and increased grassland productivity [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Its high yield and resilience to environmental stressors make it an exceptional forage species, supporting the development of grassland-based livestock farming and ecological restoration efforts. Recently, the reference genome sequence of \u003cem\u003eE. Sibiricus\u003c/em\u003e was published, providing valuable insights into its genetic basis and evolutionary history through in-depth genome and population genomic analyses [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This high-quality genomic reserach establishes a solid scientific foundation for future breeding programs and practical applications of \u003cem\u003eE. Sibiricus.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eIn this study, the \u003cem\u003eSPL\u003c/em\u003e (SQUAMOSA promoter-binding protein-like) genes were systematically exploration, identified, and analyzed in \u003cem\u003eE. sibiricus\u003c/em\u003e by using its genome as a reference. A total of 37 \u003cem\u003eEsSPL\u003c/em\u003e genes were identified, and their evolutionary relationships were examined in comparison with \u003cem\u003eSPL\u003c/em\u003e genes from various species, including \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eSolanum lycopersicum\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eSorghum bicolo\u003c/em\u003e, \u003cem\u003eZea mays\u003c/em\u003e, \u003cem\u003eBrachypodium distachyon\u003c/em\u003e and \u003cem\u003eTriticum aestivum\u003c/em\u003e. Phylogenenic analysis revealed that \u003cem\u003eE. sibiricus\u003c/em\u003e shares a closer evolutionary relationship with wheat.Additionally, chromosomal distribution, gene duplication events, cis-acting regulatory elements, gene structures, and conserved motifs of \u003cem\u003eEsSPL\u003c/em\u003e family were investigated. Notably, 16 \u003cem\u003eEsSPL\u003c/em\u003e genes were found to contain recognition sites for miR156. suggesting thier potential regulation bu this microRNA. Analysis of cis-acting regulatory elements in the promoter regions revealed an enrichment of hormone response elements, indicating their possible roles in hormone signaling pathways. Furthermore, gene expression analysis demonstrated that most \u003cem\u003eEsSPL\u003c/em\u003e genes exhibit high expression levels in seedling tissues. Specifically, \u003cem\u003eEsSPL2\u003c/em\u003e was signicantly upregulated under four different types of abiotic stress, suggesting its involvement in stress rsponses. In summary, this comprehensive analysis provided valuable insights into the \u003cem\u003eSPL\u003c/em\u003e gene family in \u003cem\u003eElymus sibiricus\u003c/em\u003e, highlighting \u003cem\u003etheir crucial roles\u003c/em\u003e in growth ,development and stress adaptation. Our findings not only establish a foundation for future functional studies of EsSPL genes but also but also contribute to a deeper understanding of the genetic characteristics and potential applications of \u003cem\u003eE. sibiricus\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Identification and chromosomal localization of \u003cem\u003eElymus sibiricus SPL\u003c/em\u003e genes\u003c/h2\u003e \u003cp\u003eIn this study, the genomic data for \u003cem\u003eE. sibiricus\u003c/em\u003e was provided by the Scientific Data (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.nature.com/articles/s41597-024-03622-4\u003c/span\u003e\u003cspan address=\"https://www.nature.com/articles/s41597-024-03622-4\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e provided on September 6th, 2024)[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The \u003cem\u003eArabidopsis AtSPL\u003c/em\u003e sequences were retrieved from the TAIR database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"http://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To identifiy \u003cem\u003eEsSPL\u003c/em\u003e genes, BLASTp analysis was performed in TBtools using \u003cem\u003eAtSPL\u003c/em\u003e sequences as queries. Additionally, conserved domain analysisi was conducted using the CDD-Search database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), while the InterPro database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/interpro/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized to screen for SBP-domain and non-SBP-domain proteins. Pfam model files were downloaded from Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro/download/Pfam/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/interpro/download/Pfam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). using these resources, TBtools confirmed that the \u003cem\u003eE.sibiricus\u003c/em\u003e SPL(\u003cem\u003eEsSPL\u003c/em\u003e) gene family contains SBP domains (PF03110). The chromosomal localization of \u003cem\u003eEsSPL\u003c/em\u003e genes was determained based on genome annotation data for \u003cem\u003eE. sibiricus.\u003c/em\u003e Additionally, The physicochemical properties of \u003cem\u003eEsSPL\u003c/em\u003e proteins were analyzed using the ExPASy protParam tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://web.expasy.org/\u003c/span\u003e\u003cspan address=\"http://web.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This analysis included parameters such as amino acid length, molecular weight (MW), isoelectric point (PI), instability index, aliphatic index, and average hydrophobicity (GRAVY).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Phylogenetic analysis of the \u003cem\u003eEsSPL\u003c/em\u003e gene family\u003c/h2\u003e \u003cp\u003eSPL amino acids sequences were obtained from multiple databases for \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eSolanum lycopersicum\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eSorghum bicolor\u003c/em\u003e, \u003cem\u003eBrachypodium distachyon\u003c/em\u003e, \u003cem\u003eTriticum: aestivum\u003c/em\u003e, and \u003cem\u003eZea mays\u003c/em\u003e. These sequences were obtained from TAIR (The \u003cem\u003eArabidopsis\u003c/em\u003e Information Resource), the Rice Genome Annotation Project, the Sol Genomics Network, and GIGADB (Genomic Data Commons for Agriculture \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.gigadb.org\u003c/span\u003e\u003cspan address=\"http://www.gigadb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.) To investigate the evolutionary relationships among the \u003cem\u003eEsSPL\u003c/em\u003e gene family members, a comprehensive phylogenetic tree was constructed. For further functional analysis, additional SPL amino acid sequences from the same species were retrieves from NCBI (National Center for Biotechnology Information \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Pear MODB (a molecular database for plant sequences \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.plantgdb.org/\u003c/span\u003e\u003cspan address=\"http://www.plantgdb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Plant TFDB (a comprehensive database for plant transcription factors \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://planttfdb.gao-lab.org/\u003c/span\u003e\u003cspan address=\"http://planttfdb.gao-lab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and TAIR. The phylogenetic tree was generated using the Neighbor-Joining (NJ) method in MEGA 11.0 software, with 1,000 bootstrap replications to ensure statistical robustness. The resulting tree was then visualized using the iTOL online platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://itol.embl.de/\u003c/span\u003e\u003cspan address=\"http://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) to provide a clear and structured representation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Gene structure and multiple sequence alignment analysis\u003c/h2\u003e \u003cp\u003eMotif analysis of the \u003cem\u003eEsSPL\u003c/em\u003e gene family proteins was conducted using the MEME tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite.org/tools/meme\u003c/span\u003e\u003cspan address=\"http://meme-suite.org/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) as described in [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], A totle of 37 motifs were identified. Additionally, the gene structures and conserved domains of \u003cem\u003eEsSPL\u003c/em\u003e family members were analyzed using TBtools to gain a comprehensive understanding of their characteristics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Gene duplication and synteny analysis of the \u003cem\u003eEsSPL\u003c/em\u003e gene family\u003c/h2\u003e \u003cp\u003eThe chromosomal location and mapping information for the \u003cem\u003eEsSPL\u003c/em\u003e gene family members were obtained from the \u003cem\u003eE. sibiricus\u003c/em\u003e genome annotation file (GFF3 format) using TBtools. This enabled the visualization of gene distribution across chromosomes, as described in Chen study[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To explore syntenic relationships, genonmic sequences of \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eTriticum aestivum\u003c/em\u003e were downloaded from the Phytozome database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Synteny analysis was then performed using the One Step MCScanX function in TBtools, comparing \u003cem\u003eE. sibiricus\u003c/em\u003e with \u003cem\u003eArabidopsis\u003c/em\u003e and wheat. The analysis parameters were set to a 'Num of Blast Hits' of 4 and an E-value threshold of 1e-10 to ensure accuracy and reliability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Identification of cis-acting regulatory elements in the promoters and prediction of protein-protein interactions\u003c/h2\u003e \u003cp\u003eBelow is a paragraph from an academic paper. Polish the writing to meet the academic style,improve the spelling, grammar, clarity, concision and overall readability. When necessary, rewrite the whole sentence. List the modified paragraphs. Furthermore, list all modification and explain the reasons to do so in markdown table. Paragraph : To investigate the potential functions and expression regulation mechanisms of the \u003cem\u003eEsSPL\u003c/em\u003e genes, the 2000 bp upstream promoter sequences of these genes were extracted and uploaded to NCBI. The PlantCARE database (accessed on September 14, 2024, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and TBtools were used for visualization [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Protein-protein interaction predictions were carried out using the online STRING database (accessed on September 14, 2024, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org/\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Interaction protein information was retrieved from UniProt (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on September 14, 2024).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Growth conditions and stress treatments of \u003cem\u003eElymus sibiricus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe materials of \u003cem\u003eElymus sibiricus\u003c/em\u003e utilized in this experiment were provided by the College of Animal Science and Veterinary Medicine at Qinghai University, China. These plant materials were cultivated within an artificial climate chamber at the Institute of Animal Science and Veterinary Medicine of the Chinese Academy of Agricultural Sciences in Beijing, China. Hydroponic seedlings of \u003cem\u003eElymus sibiricus\u003c/em\u003e were grown in a controlled environment with a photoperiod of 16 hours light (25\u0026deg;C) followed by 8 hours darkness (18\u0026deg;C), a light intensity of 250 \u0026micro;mol photons\u0026middot;m-2\u0026middot;s-1, and a relative humidity of 70%, with regular maintenance. For abiotic stress treatments, seedlings two weeks old were utilized. To investigate gene expression levels in various tissues, roots, stems, leaves, and spikes were gathered at the heading stage for expression analysis. Additionally, seeds and young roots and seedlings of \u003cem\u003eElymus sibiricus\u003c/em\u003e, two weeks old, were sampled for expression analysis as well. All samples were subjected to three biological replicates. The data was visualized using Amazing Heat Map software to generate heatmaps that offer a clear representation of the experimental results. Regarding abiotic stress treatments, two-week-old \u003cem\u003eElymus sibiricus\u003c/em\u003e seedlings were exposed to salt (200 mM NaCl), drought (20% PEG6000), abscisic acid (ABA at 0.1 mM), and gibberellin (GA at 0.1 mM). Samples were collected at various time points: 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 100 h. All experimental procedures were conducted with three biological replicates to ensure accuracy and reliability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Total RNA extraction and qPCR analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted utilizing the Trizol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer's prescribed protocol. Subsequently, genomic DNA contamination was eliminated through DNase digestion employing the services of Promega (Madison, WI, USA). The synthesis of the first-strand cDNA was accomplished using M-MLV reverse transcriptase (also provided by Promega) with 1 \u0026micro;g of the total RNA as a template. RT-qPCR analysis was performed on the CFX96 Touch\u0026trade; Real-Time PCR Detection System from Bio-Rad (Hercules, CA, USA). The PCR amplification parameters were set as: an initial denaturation at 95\u0026deg;C for 2 minutes, followed by 40 cycles of denaturation at 95\u0026deg;C for 15 seconds and annealing/extension at 60\u0026deg;C for 40 seconds. A final cycle consisted of denaturation at 95\u0026deg;C for 15 seconds and annealing/extension at 60\u0026deg;C for 15 seconds. The alfalfa actin gene was utilized as the internal reference, and relative gene expression was analyzed using the 2^-∆∆Ct methodology. All experimental procedures were repeated with three biological replicates and three technical replicates to ensure reliability. Primer design was done using Primer 5.0 software for accurate and consistent results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Data analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was conducted utilizing the one-way analysis of variance (ANOVA) within the SPSS software version 23.0. The data was presented in the form of mean values accompanied by their corresponding standard deviations. Graphs were created and thoroughly analyzed using GraphPad Prism 10.1.1 software. Any observed differences were deemed statistically significant when the P-value was below 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Genome-wide identification of the \u003cem\u003eSPL\u003c/em\u003e gene family in \u003cem\u003eElymus sibiricus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eUsing comparative genomics techniques, a total of 37 candidate genes were identified in the \u003cem\u003eE. sibiricus\u003c/em\u003e genome [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These genes were named based on their sequence homology with corresponding \u003cem\u003eArabidopsis\u003c/em\u003e SPL proteins. The key characteristics of the \u003cem\u003eEsSPL\u003c/em\u003e gene family, including TIGR loci, chromosomal locations, coding and amino acid sequence lengths, molecular weights, theoretical isoelectric points, instability indices, aliphatic indices, and the hydrophilicity, are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The amino acid lengths of \u003cem\u003eEsSPL\u003c/em\u003e proteins exhibit significant variation, ranging from 192 to 1,129 residues. Their molecular weight span from 20,156.51 to 123,837.02. The theoretical isoelectric points value range from 5.49 to 9.99, while instability indices vary between 51.57 and 72.05, indicating that these proteins maybe unstable. Furthermore, the aliphatic index varies range from 41.47 to 80.84, reflecting differences in thermostability. Importantly, the average grand hydropaphy(GRAVY) scores of \u003cem\u003eEsSPL\u003c/em\u003e proteins are negative, suggesting that they are predominantly hydrophilic.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList and detailed information of identified \u003cem\u003eSPL\u003c/em\u003e genes in \u003cem\u003eElymus sibiricus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChr\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCDS Length\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePep Length\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMolecular Weight(kDa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTheoretical pI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eInstability Index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAliphatic Index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGrand Average of Hydropathicity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr01.9142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e579\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20156.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e57.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e59.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.627\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr01.18876\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e321\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e33678.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e53.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e58.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.459\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr01.21724\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1230\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e409\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44107.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e57.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e49.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.779\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr02.9866\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e579\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20175.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e62.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e58.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.641\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr02.18548\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1386\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e461\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e47970.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e53.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e67.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.254\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr02.21396\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e407\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43930.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e57.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e48.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.779\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr03.12983\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e859\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e94207.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e53.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e79.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL8\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr04.13338\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e859\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e94063.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e51.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e80.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL9\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr05.4949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1557\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e518\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e55294.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e61.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e60.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.454\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr06.5732\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1209\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e402\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42334.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e58.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e54.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.538\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL11\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr07.7740\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2514\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e837\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e92845.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e62.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e77.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.388\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL12\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr07.9472\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e418\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43461.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e55.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e50.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.615\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr07.9649\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1129\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e123837.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e54.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e72.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.536\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL14\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr07.9826\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1095\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e364\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e38309.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e63.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e57.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.493\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL15\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr07.21378\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43488.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e60.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e50.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.562\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL16\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr07.21380\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e418\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44408.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e50.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.609\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL17\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr08.7950\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2523\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e840\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e93125.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e59.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e80.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.353\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL18\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr08.9588\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e381\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e39992.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e54.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e46.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.755\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL19\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr08.9908\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1129\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e123643.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e52.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e74.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.511\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL20\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr08.10315\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1275\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e424\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e45158.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e60.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e57.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.492\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL21\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr08.20197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43199.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e52.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e60.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.487\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL22\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr08.20224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e394\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42515.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e60.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e52.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.588\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL23\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr08.20226\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1251\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e416\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44490.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e55.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e52.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.604\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL24\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr09.13491\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e408\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42487.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e61.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e41.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.739\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL25\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr09.14379\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e429\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44773.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e63.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e57.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.378\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL26\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr09.21556\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2892\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e963\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e105347.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e54.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e78.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.311\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL27\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr10.13639\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1203\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e41905.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e60.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e42.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.705\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL28\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr10.14363\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e433\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e45246.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e61.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e55.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.371\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL29\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr10.21624\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2898\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e965\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e105583.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e53.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e78.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.301\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL30\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr11.2280\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e408\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43789.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e60.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e60.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.388\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL31\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr11.5748\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1422\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e473\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e51073.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e57.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e52.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.619\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL32\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr11.7263\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e449\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e48342.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e72.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e47.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.631\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL33\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr11.7419\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e981\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e326\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e35316.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e67.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e53.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.668\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL34\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr12.4808\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1422\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e473\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e51077.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e62.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e50.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.645\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL35\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr12.6316\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1461\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e486\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e52010.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e63.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e63.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.345\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr12.6799\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e978\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e34954.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e61.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e54.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.611\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEsSPL37\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eevm.model.Chr14.18350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e933\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e310\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e33707.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e61.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e56.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e-0.535\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Phylogenetic tree analysis of the \u003cem\u003eSPL\u003c/em\u003e gene family in \u003cem\u003eElymus sibiricus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo explore the evolutionary relationships of SPL proteins in \u003cem\u003eE. sibiricus\u003c/em\u003e, a comprehensive phylogenetic analysis was conducted using multiple model plants species, including \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eS. lycopersicum, O. sativa, S. bicolor, B. distachyon\u003c/em\u003e, \u003cem\u003eT. aestivum\u003c/em\u003e, and \u003cem\u003eZ. mays\u003c/em\u003e. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To further explore the structural complexity of \u003cem\u003eSPL\u003c/em\u003e gene family, a Neighbor-Joining (NJ) phylogenetic tree was constructed based on \u003cem\u003eSPL\u003c/em\u003e protein sequences from \u003cem\u003eE. sibiricus\u003c/em\u003e (19), \u003cem\u003eA. thaliana\u003c/em\u003e (17), \u003cem\u003eS. lycopersicum\u003c/em\u003e (15), \u003cem\u003eO. sativa\u003c/em\u003e (19), \u003cem\u003eS. bicolor\u003c/em\u003e (18), \u003cem\u003eZ. mays\u003c/em\u003e (31), \u003cem\u003eB. distachyon\u003c/em\u003e (18), and \u003cem\u003eT. aestivum\u003c/em\u003e (56). The phylogenetic analysis grouped these SPL genes into seven distinct clades, revealing evolutionary patterns among species. Notably, in Group 6, \u003cem\u003eEsSPL\u003c/em\u003e genes clustered closely with wheat \u003cem\u003eTaSPL\u003c/em\u003e genes, suggesting a strong co-evolutionary relationship between \u003cem\u003eE. sibiricus\u003c/em\u003e and \u003cem\u003eT. aestivum\u003c/em\u003e. Specifically, the homologous genes such as \u003cem\u003eEsSPL15\u003c/em\u003e, \u003cem\u003eEsSPL16\u003c/em\u003e, \u003cem\u003eEsSPL21\u003c/em\u003e, \u003cem\u003eEsSPL22\u003c/em\u003e, and \u003cem\u003eEsSPL23\u003c/em\u003e exhibited high sequence similarity with \u003cem\u003eT. aestivum TaSPL\u003c/em\u003e genes (e.g., \u003cem\u003eTaSPL\u003c/em\u003e16A, \u003cem\u003eTaSPL\u003c/em\u003e16D ), implying potential functional conservation and shared evolutionary trajectories that warrant further investigation. Furthermore, the analysis revealed a significant expanaion of the \u003cem\u003eSPL\u003c/em\u003e gene family from lower to higher plants.This expansion is accompanied by extensive gene duplication events, which likely contributed to the evolutionary success of flowering plants by enhancing their adaptability to diverse and fluctuating environmental conditions [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Gene structure analysis of the \u003cem\u003eSPL\u003c/em\u003e gene family in \u003cem\u003eElymus sibiricus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo further explore the functional diversity of SPL-associated candidate genes in \u003cem\u003eE. sibiricus\u003c/em\u003e, we analyzed their conserved motifs, domains, and gene structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Using the MEME suite for multiple sequence alignments, the sequence markers that target the SBP domain in \u003cem\u003eE. sibiricus\u003c/em\u003e were identified. This approach facilitated the comprehensive assessment of the conservation of homologous domain sequences, particularly in relation to the SBP domain. SPL proteins are structurally characterized by two zinc ion-binding motifs: C3H (Cys-Cys-Cys-His) and C2HC (Cys-Cys-His-Cys). Additionally, a Nuclear Localization Signal (NLS) overlaps with the second zinc finger structure at the carboxyl terminus of the SBP domain. Our analysis revealed that Motif 1 and Motif 2 in \u003cem\u003eE. sibiricus\u003c/em\u003e domain correspond to Zn finger motifs, both of which are present in all EsSPL proteins, highlighting a high degree of sequence conservation. Notably, \u003cem\u003eEsSPL37\u003c/em\u003e is the only excpeption, as it lacks Motif 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To investigate post-transcriptional regulation, we alighed complementary sequences of miR156 with the multiple sequence alignment of \u003cem\u003eEsSPL\u003c/em\u003e genes in (Supplementary Fig.\u0026nbsp;1). The analysis identified miR156 recognition sites in 16 out of 37 \u003cem\u003eEsSPL\u003c/em\u003e genes, acounting for 43.2% of the gene family. Interestingly, these recognition sites were consistently located within motif 7, suggesting a conserved regulatory mechaism. Beyond the SBP domain, certain EsSPL proteins exhibited additional structural features. Specially, EsSPL26 and EsSPL29 possess an extra ANKYR domain, which may imply functional diversification (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Analysis of the intron-exon structures showed that the the number of introns among \u003cem\u003eEsSPL\u003c/em\u003e genes varies from 2 to 11, with a maximum of three exons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Collectively, these findings provide critical insights into the structural conservation and functional specificity of SPL-related candidate genes in \u003cem\u003eE. sibiricus.\u003c/em\u003e The presence of conserved domains, motifs, and gene structures suggests their essential roles in various biological processes, highlighting their evolutionary significance and regulatory potential.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Chromosomal location and synteny analysis\u003c/h2\u003e \u003cp\u003eThrough mapping the open reading frames (ORFs) of all identified EsSPL genes to their respective chromosomes, the distribution of the \u003cem\u003eSPL\u003c/em\u003e gene family in \u003cem\u003eElymus sibiricus\u003c/em\u003e were determined (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The results indicated a non-uniform distribution of \u003cem\u003eEsSPL\u003c/em\u003e genes across the 14 chromosomes of \u003cem\u003eE. sibiricus\u003c/em\u003e. Specifically, chromosome 8 contained the highest number of \u003cem\u003eSPL\u003c/em\u003e genes, with 7 identified, followed by chromosomes 7 (6 genes), chromosome 11 (4 genes), and chromosome1and 2( (3 genes each). In contrast, chromosome 3, 4, 5, 6, and 14 contained only aa single \u003cem\u003eEsSPL\u003c/em\u003e gene, while chromosome 13 lacked any \u003cem\u003eEsSPL\u003c/em\u003e genes entirely. Synteny analysis within \u003cem\u003eE. sibiricus\u003c/em\u003e identified 19 gene pairs., demonstrating that members of the \u003cem\u003eEsSPL\u003c/em\u003e gene family predominantly exist in tandem. This pattern is likely a comsequence of the allopolyploid nature of \u003cem\u003eE. sibiricus\u003c/em\u003e, which has led to the retention of duplicated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To further elucidate the evolutionary relationships of the \u003cem\u003eEsSPL\u003c/em\u003e gene family, a comparative synteny map was constructed among \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eE. sibiricus\u003c/em\u003e, and \u003cem\u003eT. aestivum\u003c/em\u003e. Interestingly, \u003cem\u003eArabidopsis\u003c/em\u003e shares 6 orthologous genes pairs with \u003cem\u003eE. sibiricus\u003c/em\u003e, whereas \u003cem\u003eE. sibiricus\u003c/em\u003e shares a significantly higher number of orthologous genes pairs (25) with wheat (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eCalculation of the nonsynonymous-tosynonymous substitution rate ratio (Ka/Ks) for paralogous gene pairs revealed that all values were less than 1, Indicated that these genes are under purifying selection and have conserved functions throughout evolution (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, the estimated divergence times for both orthologous and paralogous gene pairs provide valuable evolutionary insights. The divergence time for orthologous gene pairs ranged from 37.83 to 168.38\u0026nbsp;million years ago (MYA), whereas t paralogous gene pairs diverged between36.84 and 246.61 MYA. These findings suggest that the expansion of the \u003cem\u003eSPL\u003c/em\u003e gene family in \u003cem\u003eE. sibiricus\u003c/em\u003e is likely a result of an ancient whole-genome duplication (WGD) event, followed by subsequent segmental duplications (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKa and Ks values of homologous gene pairs in \u003cem\u003eElymus sibiricus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKa/Ks\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePurify selection\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDuplication type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTime\u0026thinsp;=\u0026thinsp;Ks/2λ ( MYAa)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEsSPL1/EsSPL2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.2969\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9044\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSegmental\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e74.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEsSPL2/EsSPL9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3979\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.712\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.5588\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSegmental\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e58.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEsSPL3/EsSPL15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3754\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.4591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSegmental\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e67.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEsSPL4/EsSPL21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.4454\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8534\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.5219\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSegmental\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e70.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEsSPL12/EsSPL20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3821\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.0634\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3593\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSegmental\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e87.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEsSPL15/EsSPL25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.337\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3652\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSegmental\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e76.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEsSPL15/EsSPL33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.4147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8941\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.4638\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSegmental\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e73.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEsSPL15/EsSPL36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.4154\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.6889\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.6030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSegmental\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e56.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEsSPL16/EsSPL20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3687\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.984\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSegmental\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e246.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEsSPL20/EsSPL30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.1366\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5716\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSegmental\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e47.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEsSPL22/EsSPL33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.4147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.9751\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.4253\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSegmental\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e80.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEsSPL23/EsSPL36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.4277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8875\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.4819\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSegmental\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e73.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEsSPL24/EsSPL30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.5296\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.4342\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2176\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSegmental\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e201.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEsSPL30/EsSPL37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.2636\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.4578\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.5758\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSegmental\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e37.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Protein-protein interaction network analysis of the \u003cem\u003eSPL\u003c/em\u003e gene family in \u003cem\u003eE. sibiricus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eA protein-protein interaction (PPI) network was constructed and analyzed using STRING network modeling technology, revealing key topological features. The analysis identified 37 SPL family proteins predicted to interac with a diverse range of proteins, suggesting their potential s t roles in pathways related to plant hormone signaling, environmental stress responses, as well as growth and developmental processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Notably, homologous proteins exhibit similar interaction patterns, as observed in EsSPL1 and EsSPL4, EsSPL2 and EsSPL16, and various other EsSPL proteins pairs. This indicates that these protein pairs may share conserved interaction domains, highlighting substantial connectivity within the \u003cem\u003eEsSPL\u003c/em\u003e network and its intercations with other proteins. Additionally, SOC1 and TOE2 frequently appear in these interaction networks. Previous studies have demonstrated that SOC1 gene functions as a central integrator of flowering signals, intergrating inputs from multiple pathways -including photoperiod, autonomous, gibberellin, vernalization, and age-dependent pathways- to regulate flowering time. Similarly, cytokinien modifies the transition from vegetative to reproductive growth via miR172 and its target genes TOE1 and TOE2[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This analysis provides deeper understanding of the functional regulation of interacting proteins within the \u003cem\u003eSPL\u003c/em\u003e gene family,offering a comprehensive perspective on their intricate interactions and roles in biological processes.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.6 Identification of cis-acting regulatory elements in the promoters of the\u003c/b\u003e \u003cb\u003eSPL\u003c/b\u003e \u003cb\u003egene family in\u003c/b\u003e \u003cb\u003eElymus sibiricus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUsing Plant CARE to analyze the promoter regions of \u003cem\u003eEsSPL\u003c/em\u003e genes, a total of 966 cis-acting regulatory elements were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Among them, 369 cis-acting regulatory elements were associated with hormone response regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Four hormone-responsive motifs were detected, including methyl jasmonate response elements (CGTCA-motif), salicylic acid response elements (TCA-element), gibberellin response elements (P-box), and abscisic acid (ABA) response elements (ABRE). Additionally, various stress-responsive elements, were identified such as drought-related regulatory elements (MBS), low-temperature response elements (LTR), anaerobic response elements (ARE), and defense and stress response elements (TC-rich) etc. These cis-acting regulatory elements were classified into four major categories: environmental stress response elements, hormone response elements, development-related elements, and light response elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Among the environmental stress response elements, anaerobic response elements were the most abundant, accounting for 33.2% of the total. Within the hormone response element, ABRE motif were the most prevalent, constituting 64.8%, followed by CGTCA-motif elements at 27.4%. Among the five identified development-related elements, the CAT-box element was the most frequent(( 40.6%), followed by the O2-site element (29.2%). For light light-responsive elements, the G-box element was the most common(52.8%), followed by the \u003cem\u003eSPL\u003c/em\u003e element ( 21.2%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Notably, the distribution of cis-acting regulatory elements varied among different EsSPL genes. \u003cem\u003eEsSPL20\u003c/em\u003e exhibited a higher proportion of hormone and light response cis-acting regulatory elements, whereas \u003cem\u003eEsSPL3\u003c/em\u003e and \u003cem\u003eEsSPL6\u003c/em\u003e contained a greater number of hormone response elements. In cntrast\u003cem\u003eEsSPL28\u003c/em\u003e harbored more environmental stress response elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Tissue-specific expression of \u003cem\u003eSPL\u003c/em\u003e-Related candidate genes in \u003cem\u003eE. sibiricus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo investigate the potential functions of \u003cem\u003eSPL\u003c/em\u003e-related candidate genes in different tissues of \u003cem\u003eE. sibiricus\u003c/em\u003e and determine whether these genes serve broad housekeeping roles or paerticipate in tissue-specific regulatory mechanisms, a tissue-specific expression analysis was performed(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The results revealed that \u003cem\u003eEsSPL33\u003c/em\u003e exhibits strong expression in root tissues, while \u003cem\u003eEsSPL15\u003c/em\u003e and \u003cem\u003eEsSPL16\u003c/em\u003e are highly expressed in stem. Similarly, \u003cem\u003eEsSPL4\u003c/em\u003e demonstrates elevated expression in leaves. Several genes, including \u003cem\u003eEsSPL8, EsSPL10, EsSPL25, EsSPL28, EsSPL34\u003c/em\u003e, and \u003cem\u003eEsSPL36\u003c/em\u003e, displayelevated expression levels in spike tissues. Notably, a large subset of \u003cem\u003eEsSPL\u003c/em\u003e genes (\u003cem\u003eEsSPL1, EsSPL2, EsSPL3, EsSPL6, EsSPL7, EsSPL13, EsSPL14, EsSPL17, EsSPL18, EsSPL21, EsSPL24, EsSPL26, EsSPL29, EsSPL31, EsSPL35\u003c/em\u003e, and \u003cem\u003eEsSPL37\u003c/em\u003e) showed predominant expression in seedlings. Additionally, \u003cem\u003eEsSPL9, EsSPL23, EsSPL27\u003c/em\u003e, and \u003cem\u003eEsSPL35\u003c/em\u003e exhibit strong expression in young roots, whereas \u003cem\u003eEsSPL5\u003c/em\u003e, \u003cem\u003eEsSPL11, EsSPL12, EsSPL19, EsSPL20, EsSPL22, EsSPL30\u003c/em\u003e, and \u003cem\u003eEsSPL32\u003c/em\u003e are highly expressed in seeds. It is worth noting that, similar to \u003cem\u003eSetaria italic\u003c/em\u003e (foxtail millet), most SPL gene family members in \u003cem\u003eE. sibiricus\u003c/em\u003e exhibit high expression levels in seedlings, suggesting a conserved regulatory role during plant early development stages.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.8 Expression of\u003c/b\u003e \u003cb\u003eSPL\u003c/b\u003e\u003cb\u003e-related candidate genes in\u003c/b\u003e \u003cb\u003eE. sibiricus\u003c/b\u003e \u003cb\u003ein response to salt, drought, ABA, and GA stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess the role of \u003cem\u003eSPL\u003c/em\u003e-related candidate genes in the stress response of \u003cem\u003eE. sibiricus\u003c/em\u003e, their expression levels were analyzed following exposure to abiotic stress conditions. Two-week-old plants were subjected to hydroponic treatments, 200 mM NaCl (salt stress), 20% PEG6000(drought stress), 0.1 mM abscisic acid (ABA )), and 0.1 mM gibberellin (GA).Treatments were administered for up to 100 hours, with samples collected at intervals of 3, 6, 12, 24, 48, 72, and 100 hours. For each treatment and time point, three plants were carefully harvested, and gene expression ws quatified using three biological replicates to ensure accuracy Under salt stress conditions, most SPL genes exhibited a downregulation trend over time. Specifically, \u003cem\u003eEsSPL5\u003c/em\u003e, \u003cem\u003eEsSPL6, EsSPL13, EsSPL16, EsSPL19, EsSPL25, EsSPL26, EsSPL27\u003c/em\u003e, and \u003cem\u003eEsSPL28\u003c/em\u003e were significantly downregulated compared to their pre-treatment expression levels. In contrast, \u003cem\u003eEsSPL2, EsSPL7, EsSPL10, EsSPL11, EsSPL14, EsSPL17, EsSPL20, EsSPL21, EsSPL23, EsSPL33\u003c/em\u003e, and \u003cem\u003eEsSPL34\u003c/em\u003e exhibited an initial increase followed by a decline. Notably, \u003cem\u003eEsSPL4\u003c/em\u003e and \u003cem\u003eEsSPL9\u003c/em\u003e genes showed sustained upregulation, with expression levels increasing 5.45-fold and 6.55-fold, respectively, compared to pre-treatment levels ( Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These findings suggested that \u003cem\u003eEsSPL4\u003c/em\u003e and \u003cem\u003eEsSPL9\u003c/em\u003emay play significantly roles in negative regulation under salt stress and could serve as potential targets for future studies on salt stress adaptation. Similarly, under drought stress conditions, many \u003cem\u003eSPL\u003c/em\u003e genes exhibited an initial upregulation followed by a gradual decrease over time. Specifically, \u003cem\u003eEsSPL2, EsSPL3, EsSPL10, EsSPL11, EsSPL12, EsSPL13, EsSPL14 and EsSPL1\u003c/em\u003e8, were significantly upregulated. Among them, \u003cem\u003eEsSPL8\u003c/em\u003e and \u003cem\u003eEsSPL16\u003c/em\u003e genes demonstrated the most pronounced increases, with expression levels rising 5.66-fold and 4.75-fold, respectively. compared to pre-treatment levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results indicated that \u003cem\u003eSPL\u003c/em\u003e gene family members play a crucial role in drought stress regulation, particularly \u003cem\u003eEsSPL8\u003c/em\u003e and \u003cem\u003eEsSPL16\u003c/em\u003e, which may contribute to osmotic stress adaptation and root development processes.\u003c/p\u003e \u003cp\u003eUnder ABA treatment, the expression levels of \u003cem\u003eEsSPL17, EsSPL22\u003c/em\u003e, and \u003cem\u003eEsSPL36\u003c/em\u003e gradually decreased over time, indicating significant downregulation compared to pre-treatment levels. In contrast, \u003cem\u003eEsSPL6, EsSPL11, EsSPL12, EsSPL15, EsSPL27\u003c/em\u003e, \u003cem\u003eEsSPL30\u003c/em\u003e, \u003cem\u003eEsSPL31, EsSPL33\u003c/em\u003e, and \u003cem\u003eEsSPL34\u003c/em\u003e exhibited an initial upregulation followed by a subaequent decline (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Specifically, \u003cem\u003eEsSPL2\u003c/em\u003e displayed a substantial regulation of 5.52-fold compared to pre-treatment levels. Interestingly, the upregulation of \u003cem\u003eEsSPL2, EsSPL11, EsSPL12\u003c/em\u003e, and \u003cem\u003eEsSPL27\u003c/em\u003e under ABA treatment was consistent with their expression patterns observed in the drought treatment, suggesting that \u003cem\u003eSPL\u003c/em\u003e transcription factors may play a crucial role in ABA-mediated stress responses GA (gibberellin) regulates various plant growth and developmental processes, including seed germination, flower organ development, etc. Under GA treatment, the expression levels of several genes including \u003cem\u003eEsSPL7, EsSPL8, EsSPL9, EsSPL10, EsSPL15, EsSPL16, EsSPL19, EsSPL20, EsSPL22, EsSPL25, EsSPL27, EsSPL32, EsSPL34, EsSPL36\u003c/em\u003e, and \u003cem\u003eEsSPL37\u003c/em\u003e initially increased before gradually declining, indicating a significant upregulation in comparison to their pre-treatment levels. Specifically, \u003cem\u003eEsSPL2, EsSPL14\u003c/em\u003e, and \u003cem\u003eEsSPL17\u003c/em\u003e exhibited the most pronounced increases, with expression levels rising 3.92-fold, and 5.01-fold, and 6.31-fold, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These findings suggested that \u003cem\u003eSPL\u003c/em\u003e might be involved in gibberellin signaling transduction[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], further reinforcing their regulatory roles in plant growth and stress adaptation .\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, a total of 37 \u003cem\u003eSPL\u003c/em\u003e-related candidate genes were identified in the genome of \u003cem\u003eE. sibiricus\u003c/em\u003e andcategorized into eight distinct groups. Comparative analysis with other plant species reveals a diverse gene distribution, including 17 SPL genes from \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], 15 from tomato [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], 17 from sorghum [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], 19 from rice [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], and a notably larger count of 31 from maize [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Evolutionary analysis, gene structure examination, and sequence alignment demonstrated classification patterns in \u003cem\u003eE. sibiricus\u003c/em\u003e,similar to those observed in other species. All \u003cem\u003eEsSPL\u003c/em\u003e genes contained the SBP domain, while EsSPL26 and \u003cem\u003eEsSPL29\u003c/em\u003e also possessed an Ankyrin domain, consistent with previous findings on the SPL gene family structure in lychee [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGene duplication is a key driver of genetic evolution [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In \u003cem\u003eE. sibiricus\u003c/em\u003e, an evolutionary pattern similar to that of other plants was observed. While most EsSPL genes exist as single-copy genes, several multi-copy genes were identified, including \u003cem\u003eEsSPL1, EsSPL4, EsSPL7\u003c/em\u003e, and \u003cem\u003eEsSPL8\u003c/em\u003e,as well as \u003cem\u003eEsSPL12, EsSPL16, EsSPL20, EsSPL24\u003c/em\u003e, and \u003cem\u003eEsSPL30\u003c/em\u003e, which are also found across across various species. Notably, duplicated SPL genes in \u003cem\u003eE. sibiricus\u003c/em\u003e are distributed across different chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e), reflecting their complex evolutionary history. Most SPL regulatory genes appear to have originated from whole-genome duplication (WGD) events, while genes such as \u003cem\u003eEsSPL4, EsSPL8, EsSPL16, EsSPL20\u003c/em\u003e, and \u003cem\u003eEsSPL30\u003c/em\u003e likely arose through segmental duplications (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These findings suggested that the expansion of \u003cem\u003eSPL\u003c/em\u003e genes is predominantly driven by WGD and segmental duplication. Moreover, interspecies collinearity analysis revealed that 67.57% of Es\u003cem\u003eSPL\u003c/em\u003e genes have orthologs in wheat; whereas only 16.22% share orthology with \u003cem\u003eArabidopsis\u003c/em\u003e, highlighting the increasing functional divergence of SPL genes between monocots and dicots. To further explore the evolutionary dynamics of EsSPL genes,, synonymous (Ks) and and nonsynonymous (Ka) substitution rates were calculated for paralogous gene pairs. The Ka/Ks ratio, an indicator of selective pressure [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] was examined: Ka/Ks ratio\u0026gt; 1 indicates positive selection, Ka/Ks\u0026thinsp;=\u0026thinsp;1 suggests neutral selection, Ka/Ks\u0026lt; 1 indicates purifying selection. The results showed an average Ka/Ks ratio of 0.41 for paralogous \u003cem\u003eEsSPL\u003c/em\u003e gene pairs in \u003cem\u003eE. sibiricus\u003c/em\u003e, indicating that the \u003cem\u003eEsSPL\u003c/em\u003e genes have undergone purifying selection and have been subject to strong evolutionary constrains.\u003c/p\u003e \u003cp\u003eIn plants, the exon/intron structure of the \u003cem\u003eSPL\u003c/em\u003e gene exhibits low conservation withexon numbers varying significantly. In E. sibiricus, EsSPL genes contain 2 to 11 exons(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e), a range comparable to that in chickpea (2\u0026ndash;10 exons) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], sunflower (2\u0026ndash;11 exons) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], and lychee (2\u0026ndash;10 exons) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Interestingly, homologous EsSPL often often differ in exon count, such as \u003cem\u003eEsSPL32\u003c/em\u003e (3 exons) and \u003cem\u003eEsSPL33\u003c/em\u003e (6 exons), suggesting that exon gain or less has contributed to structural variations during evolution. Despite similar motif sequneces and arrangements among EsSPL genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e), substantial motif diversity was observed. Motif 3 and Motif 6 are exclusive to the fifth evolutionary group,, while Motif 4 and Motif 5 are unique to the sixth group. Additionally, Motif 8 is found only in \u003cem\u003eEsSPL14, EsSPL20, EsSPL25, EsSPL28, EsSPL30\u003c/em\u003e, and \u003cem\u003eEsSPL37\u003c/em\u003e. Conversely, the highly conserved Motifs 1 and 2, present in the core regions of EsSPL proteins, likely play a crucial role in their function as transcription factors [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Consequently, the structural diversity of EsSPL proteins is closely linked to their functional divergence, with additional motifs contributing to their specialized roles in \u003cem\u003eE. sibiricus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThis study analyzes the promoters of \u003cem\u003eSPL-\u003c/em\u003erelated candidate gene, identifying 19 cis-acting regulatory elements associated with environmental stress, plant growth, development, hormone response, and light response (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Universally prevalent elements include the hypoxia-specific response element (ABRE), linked to meristematic cell division and proliferation (CAT box), light-responsive elements (MRE, GT1-motif) (Hernandez-Garcia and Finer, 2014). Specifically, ABRE and CAT- box elements correlate with meristematic tissue development and hypoxic stress responses [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].Research by Weits et al. [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and Shukla et al. [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] highlighted the significance of apical meristem development under low oxygen conditions, essential for initiating new leaf formation. Moreover, hypoxia affects lateral root primordia by inhibiting the degradation of key regulatory proteins. Under such conditions, light acts as a signaling factor, activating stem cells through CK signaling and metabolic pathways [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The abundance of developmental elements(ABRE ,CAT-box) in SPL promoters suggests their involvement in plant hormones signalling [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], particularly in response to GA, which influences plant height and morphology. Furthermore, key cis-acting regulatory elements (GT-1-motif, CAT \u0026ndash;box,GATAT-motif) have been identified in \u003cem\u003eE. sibiricus SPL\u003c/em\u003e gene promoters[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. SPL transcription factors regulate flowering in in a tandem manner \u003cem\u003eElymus sibiricus\u003c/em\u003e and serve as a target of miR156. Overexpression of OsmiR156b and OsmiR156h in previous studies resulted in dwarfism, reduced inflorescence size, and delayed flowering, emphasizing the pivotal role of miR156-SPL regulation in embryonic development [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Given the importance of SPL expression in flowering, future studies should focus on the functional validation of these cis-acting regulatory elements, exploring SPL interactions touncover novel insights into plant growth, morphology, and developmental processes.\u003c/p\u003e \u003cp\u003eIn organisms, gene expression precedes and is essential for gene function, with expression patterns intricately linked to gene functions [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The \u003cem\u003eSPL\u003c/em\u003e gene family is widely involved in plant growth and development, playing a key role in regulating traits from seedlings to maturity [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eAtSPL10\u003c/em\u003e eliminates de novo shoot regeneration capacity by derepressing the attenuation of cytokinin response in the primary root [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In \u003cem\u003eRhododendron molle\u003c/em\u003e, \u003cem\u003eRmSPL1/7/8/12/13\u003c/em\u003e exhibit significantly divergent expression, suggesting their essential roles in regulating floral bud differentiation [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. \u003cem\u003eTaSPL6B\u003c/em\u003e recruits \u003cem\u003eTaD53\u003c/em\u003e and \u003cem\u003eTaSPL3\u003c/em\u003e to integrate light and strigolactone signaling pathways, thereby suppressing the key tillering inhibitor \u003cem\u003eTaTB1\u003c/em\u003e and enhancing \u003cem\u003eTaD53\u003c/em\u003e-mediated inhibition of \u003cem\u003eTaSPL3\u003c/em\u003e in \u003cem\u003eT. aestivum\u003c/em\u003e [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Similarly, \u003cem\u003eSPL\u003c/em\u003e genes also play crucial roles in abiotic stress responses. This study demonstrates that under salt stress, \u003cem\u003eEsSPL4\u003c/em\u003e and \u003cem\u003eEsSPL9\u003c/em\u003e exhibit sustained upregulation and likely function as important negative regulators, suggesting their potential as key targets for future research on salt stress adaptation and genetic enhancement strategies. In studies on salt tolerance in \u003cem\u003eCodonopsis pilosula\u003c/em\u003e, \u003cem\u003eCpSPL5\u003c/em\u003e and \u003cem\u003eCpSPL8\u003c/em\u003e were identified as negative regulators of salt stress resistance, indicating that members of the \u003cem\u003eSPL\u003c/em\u003e gene family are involved in the plant SOS pathway [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. In \u003cem\u003eArachis hypogaea\u003c/em\u003e (peanut) salt tolerance studies, \u003cem\u003eAhSPL5\u003c/em\u003e, \u003cem\u003eAhSPL16\u003c/em\u003e, \u003cem\u003eAhSPL25\u003c/em\u003e, and \u003cem\u003eAhSPL36\u003c/em\u003e were found to be upregulated under both drought and salinity conditions [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. In this study, \u003cem\u003eEsSPL8\u003c/em\u003e and \u003cem\u003eEsSPL16\u003c/em\u003e showed the most significant unregulation under drought stress. However, no consistent expression patterns were observed between salt and drought treatments, which might be attributed to differences in stress application conditions. In \u003cem\u003eMangifera indica\u003c/em\u003e (mango) stress resistance studies, overexpression of \u003cem\u003eMiSPL3a\u003c/em\u003e and \u003cem\u003eMiSPL3b\u003c/em\u003e in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e conferred enhanced drought tolerance and ABA-induced stress resistance [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Under ABA treatment, \u003cem\u003eEsSPL2\u003c/em\u003e, \u003cem\u003eEsSPL11\u003c/em\u003e, \u003cem\u003eEsSPL12\u003c/em\u003e, and \u003cem\u003eEsSPL27\u003c/em\u003e displayed expression patterns consistent with those observed under drought stress, suggesting that SPL transcription factors play pivotal roles in both drought and ABA-mediated stress responses. Under GA treatment, \u003cem\u003eEsSPL2\u003c/em\u003e, \u003cem\u003eEsSPL14\u003c/em\u003e, and \u003cem\u003eEsSPL17\u003c/em\u003e exhibited the most pronounced upregulation, indicating a potential role in gibberellin signal transduction and further emphasizing their regulatory functions in plant growth and stress adaptation [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Both GA and SPL genes regulate the key flowering integrators such as LFY and SOC1. GA promotes flowering by alleviating DELLA-mediated repression of these genes, while SPL transcription factors directly activate their transcription. This synergistic regulatory mechanism enables plants to fine-tune flowering time in response to internal developmental cues and external environmental signals, thereby ensuring reproductive success.\u003c/p\u003e \u003cp\u003eBased on these fondings, we propose that the \u003cem\u003eSPL\u003c/em\u003e gene family plays a crucial role in \u003cem\u003eE. sibiricus\u003c/em\u003e abiotic stress resistance. Specifically, \u003cem\u003eEsSPL2, EsSPL4, EsSPL8, EsSPL9, EsSPL14, EsSPL16\u003c/em\u003e, and \u003cem\u003eEsSPL17\u003c/em\u003e may function as keymediators of stress tolerance. Notably, \u003cem\u003eEsSPL2\u003c/em\u003e gene consistently exhibited high expression across all stress treatments compared to the control (CK), under the four stress conditions examined, the demonstrates compared to the, indicating its potential as a key regulator in stress resistance mechanisms in \u003cem\u003eE. sibiricus\u003c/em\u003e .\u003cem\u003eE. sibiricus\u003c/em\u003e may utilize \u003cem\u003eEsSPL2\u003c/em\u003e for direct stress resistance or as a regulatory hub influencing other stress-responsive genes. Determining whether \u003cem\u003eEsSPL2\u003c/em\u003e functions through direct expression or indirect regulatory mechanisms remains a critical avenue for future investigation.\u003c/p\u003e"},{"header":"5. conclusion","content":"\u003cp\u003eThis study represents the first genome-wide investigation of the SPL gene family in \u003cem\u003eE. sibiricus\u003c/em\u003e, identifying 37 EsSPL members primarily expanded through segmental duplications. Regulatory analysis revealed the presence of hormone and stress-responsive cis-acting elements in EsSPL promoters, while protein interaction predictions implicated SOC1 and TOE2 as key interacting partners. Expression profiling highlighted tissue-specific expression patterns in seedlings, with \u003cem\u003eEsSPL2\u003c/em\u003e exhibiting both constitutive expression and significant induction under four abiotic stresses. In summary, this research enhances the understanding of the genetic evolution and biological functions of the EsSPL gene family, providing a foundation for future functional characterization and application in stress adaptation studied.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSPLSquamosa promoter-binding protein-like\u003c/p\u003e\n\u003cp\u003eIPA1 \u0026nbsp;Ideal Plant Architecture 1\u003c/p\u003e\n\u003cp\u003eMW \u0026nbsp;Molecular weight\u003c/p\u003e\n\u003cp\u003ePI \u0026nbsp;isoelectric point\u003c/p\u003e\n\u003cp\u003eABA \u0026nbsp;abscisic acid\u003c/p\u003e\n\u003cp\u003eGA \u0026nbsp;gibberellin\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies involving human participants or animals performed by the authors. These methods were carried out by relevant guidelines and regulations. \u003cem\u003eElymus sibiricus\u003c/em\u003e used in this study is a prevalent wild species in the Qinghai area. Based on international standards, all the experimental research and field studies on plants, including the collection of plant material were approved by Institute of Animal Sciences. These materials are stored in Institute of Animal Sciences of Chinese Academy of Agricultural Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome sequences of \u003cem\u003eA. thaliana, S. lycopersicum, O. sativa, S. bicolor, Z. mays, B. distachyon, and T. aestivum\u003c/em\u003e were downloaded from Phytozome database (https://phytozome-next.jgi.doe.gov/). The datasets supporting the results of this article are included in the article and Additional files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Program of China (2023YFD1200303), the earmarked fund for China Agriculture Research System(CARS-34), the National Crop Germplasm Resources Center (NCGRC-63), and Xinjiang Rural Key Talent Training Project (2022SNGGGCC019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eperformed the experiments with the help of D.Y., L.M., X.M.,J.T., X.M., K.X., and W.L. analyzed the sequencing data; X.W., J.T., D.Y., Z.J., W.L., M.H., and W.L. designed the experiments, interpreted the results, and wrote the manuscript. All authors read and approved of the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all our colleagues for providing useful discussions and technical assistance. We are very grateful to the editor and reviewers for critically evaluating the manuscript and providing constructive comments for its improvement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFan E, Liu C, Wang Z, Wang S, Ma W, Lu N, Liu Y, Fu P, Wang R, Lv S, Qu G, Wang J. Genome-wide identification and expression analysis of the SQUAMOSA promoter-binding protein-like (SPL) transcription factor family in \u003cem\u003eCatalpabungei\u003c/em\u003e. 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BMC Genomics. 2015;16:787.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu M, Sun W, Ma Z, Huang L, Wu Q, Tang Z, Bu T, Li C, Chen H. Genome-wide identification of the SPL gene family in Tartary Buckwheat (\u003cem\u003eFagopyrum tataricum\u003c/em\u003e) and expression analysis during fruit development stages. BMC Plant Biol. 2019;19(1):299.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu D, Geng X, Zeng F, Xu S, Peng J. Identification and expression analysis of the SPL gene family during flower bud differentiation in Rhododendron molle. Genes Genomics. 2025;47(2):171\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong F, Song J, Zhang H, Zhang J, Chen Y, Zhou X, Li Y, Ge S, Liu Y. \u003cem\u003eTaSPL6B\u003c/em\u003e, a member of the Squamosa promoter binding protein-like family, regulates shoot branching and florescence in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. BMC Plant Biol. 2024;24(1):708.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Q, Yang Q, Dong S, Fu F, Xin Y, Kang H, Wu Y, Cao X. Transcription factors \u003cem\u003eCpSPL5\u003c/em\u003e and \u003cem\u003eCpSPL8\u003c/em\u003e negatively regulate salt tolerance in \u003cem\u003eCodonopsis pilosula\u003c/em\u003e by inhibiting SOS pathway. Plant J. 2025;121(2):e17205.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun X, Zhang L, Xu W, Zheng J, Yan M, Zhao M, Wang X, Yin Y. A Comprehensive analysis of the peanut SQUAMOSA promoter binding protein-like gene family and how \u003cem\u003eAhSPL5\u003c/em\u003e enhances salt Tolerance in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e. Plants (Basel). 2024 Apr 93(8):1057.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu J, Li Y, Zhang Y, Xia L, Hu W, Huang X, Li K, He X, Luo C. Overexpression of \u003cem\u003eMiSPL3a\u003c/em\u003e and \u003cem\u003eMiSPL3b\u003c/em\u003e confers early flowering and stress tolerance in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Int J Biol Macromol. 2024;262(Pt 1):129913.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu S, Galv\u0026atilde;o VC, Zhang YC, Horrer D, Zhang TQ, Hao YH, Feng YQ, Wang S, Schmid M, Wang JW. Gibberellin regulates the \u003cem\u003eArabidopsis\u003c/em\u003e floral transition through miR156-targeted SQUAMOSA promoter binding-like transcription factors. Plant Cell. 2012;24(8):3320\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\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":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Elymus sibiricus, SPL gene family, evolutionary relationships, gene expression, abiotic stress","lastPublishedDoi":"10.21203/rs.3.rs-6304174/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6304174/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003e\u003cem\u003eElymus sibiricus\u003c/em\u003e is widely utilized for establishing of high-yield artificial grasslands due to its remarkale productivity and strong resistance to environmental stresses, making it an excellent forage species. SPL transcription factors play a pivotal role in regulating plant growth, development, \u0026nbsp;and responses to abiotic stress. Although the \u003cem\u003eSPL\u003c/em\u003e gene family has been identified in many plant species, its presence and function in \u003cem\u003eElymus sibiricus\u003c/em\u003e remain largely unexplored.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResult:\u003c/strong\u003eThis study presents a comprehensive genome-wide identification and analysis of the \u003cem\u003eSPL\u003c/em\u003e gene family in E. \u003cem\u003esibiricus\u003c/em\u003e. A total of 37 \u003cem\u003eEsSPL\u003c/em\u003e genes were successfully identified. Their chromosomal distribution, gene structure, conserved motifs, cis-acting regulatory elements, and evolutionary relationships were analyzed. Protein-protein interaction network analysis predicted that SOC1 and TOE2 are the primary interacting proteins. Most \u003cem\u003eEsSPL\u003c/em\u003e genes exhibited high expression levels in seedling tissues. Additionally, analysis of abiotic stress responses revealed that the expression of multiple \u003cem\u003eEsSPL\u003c/em\u003e genes were altered under salt, drought, ABA, and GA treatments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThrough sequence homology analyses, 37 SPL genes were identified in E. \u003cem\u003esibiricus\u003c/em\u003e. Most \u003cem\u003eSPL\u003c/em\u003e family members exhibited high expression levels in seedling tissues, with \u003cem\u003eEsSPL2\u003c/em\u003e specifically upregulated under four distinct abiotic stress conditions. These findings provide a foundation for understanding the genetic evolution and biological functions of the SPL gene family in \u003cem\u003eE.\u003c/em\u003e \u003cem\u003esibiricus\u003c/em\u003e, offering valuable insights for future research and breeding efforts.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification and expression analysis of the SPL transcription factor family and its response to abiotic stress in Elymus sibiricus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-12 10:32:10","doi":"10.21203/rs.3.rs-6304174/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision 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