Guoliang Li, Jianting Chu, Max Haupt, Yong Jiang, Jochen C. Reif
{"title":"Genetic Dissection of Wheat Heterosis Identifies Candidate Genes in Biparental and Diverse Hybrid Populations","authors":"Guoliang Li, Jianting Chu, Max Haupt, Yong Jiang, Jochen C. Reif","doi":"10.1111/pbi.70748","DOIUrl":"https://doi.org/10.1111/pbi.70748","url":null,"abstract":"Understanding how numerous heterotic quantitative trait loci (hQTL) shape heterosis and shifting from hQTL to candidate genes are central challenges. To address these challenges, we performed a systematic, genome‐wide mapping in a biparental population and a population of diverse hybrids. Leveraging both a triple testcross and immortalized F <jats:sub>2</jats:sub> designs, we examined the genetic basis of the heterosis of the wheat hybrid <jats:italic>Piko</jats:italic> × <jats:italic>Hermann</jats:italic> . We detected a major hQTL on chromosome 4B, primarily driven by epistasis. This region contains the <jats:italic>Green Revolution</jats:italic> gene <jats:italic>Rht‐B1</jats:italic> as the primary candidate gene. Then, we used a population of ~6000 wheat hybrids, derived from crosses between diverse Central European inbred lines, to perform a one‐dimensional scan for hQTL. This scan identified 174 hQTL for grain yield, 70 for heading date, and 166 for plant height. Further dissection of these hQTL revealed that epistatic interactions predominantly contribute to heterosis. We discovered an epistatic hub at the distal end of chromosome 4A coinciding with an alien introgression region from emmer wheat. A data‐driven, integrative analysis combining high‐resolution SNP data, sequence variant annotation, and hQTL signals from the population uncovered <jats:italic>TraesCS7B03G1341000</jats:italic> as the candidate gene underlying grain yield heterosis. Our findings deepen the understanding of the genetic architecture of heterosis in wheat by shifting from hQTL to candidate genes and provide insights into their application in hybrid wheat breeding.","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"136 1","pages":""},"PeriodicalIF":13.8,"publicationDate":"2026-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148858880","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mei Guan, Chao Xie, Yang Xue, Juan Lu, Qinhong Jiang, Meizhen Yin, Shuo Yan, Jie Shen
{"title":"Interactional Endocytosis and Transmembrane Transport Promote Cellular Internalization of Nano-Delivered RNA Drugs for Efficient Control of Crop Diseases","authors":"Mei Guan, Chao Xie, Yang Xue, Juan Lu, Qinhong Jiang, Meizhen Yin, Shuo Yan, Jie Shen","doi":"10.1111/pbi.70689","DOIUrl":"10.1111/pbi.70689","url":null,"abstract":"<p><i>Botrytis cinerea</i> is a widespread plant pathogenic ascomycete that causes grey mould in over 1400 species and impacts global crop yields. Double-stranded RNA (dsRNA)-induced gene silencing is a promising technology for pest control, but efficient delivery remains a major challenge. This work presents a star polycation (SPc)-based nano-delivery platform that enhances dsRNA internalization and enables effective control of <i>B. cinerea</i>. SPc assembles with dsRNA via electrostatic interactions, hydrogen bonding and Van der Waals forces, forming stable nanoscale complexes. SPc facilitates dsRNA uptake into fungal mycelia by 2.19-fold, primarily via two activated routes: endocytosis and transmembrane transport, which increase vesicle and particle numbers by 3.74- and 1.87-fold, respectively. Six genes (<i>pkc</i>, <i>ypt10</i>, <i>pil1</i>, <i>mfs1</i>, <i>mfs2</i> and <i>mfs3</i>) in these two routes play crucial roles in the delivery process. RNA interference, chemical inhibition and mutation experiments demonstrate that the two routes interact to optimize the cellular uptake of SPc-loaded dsRNA. Finally, a high-efficiency RNA fungicide is developed, comparable to commercial fungicide in protecting cucumber leaves and tomato fruits. This study reveals the synergistic mechanism of nanocarrier-mediated gene delivery and identifies key genes, supporting the design and application of RNA pesticides.</p>","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"24 9","pages":"5152-5171"},"PeriodicalIF":12.8,"publicationDate":"2026-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398849/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148051495","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ming Luo, Aihua Wang, Soma Chakraborty, Shyh Yin Low, Saeid Babaei, Jian Chen, Jianping Zhang, Dhara Bhatt, Brenton Brooks, Oadi Matny, Narayana Upadhyaya, Rohit Mago, Melania Figueroa, Evans Lagudah, Peter Dodds, Brian Steffenson, Michael Ayliffe
{"title":"No Cost of Resistance in Wheat Gene Stack Lines Containing 10 Stem Rust Resistance Transgenes","authors":"Ming Luo, Aihua Wang, Soma Chakraborty, Shyh Yin Low, Saeid Babaei, Jian Chen, Jianping Zhang, Dhara Bhatt, Brenton Brooks, Oadi Matny, Narayana Upadhyaya, Rohit Mago, Melania Figueroa, Evans Lagudah, Peter Dodds, Brian Steffenson, Michael Ayliffe","doi":"10.1111/pbi.70692","DOIUrl":"10.1111/pbi.70692","url":null,"abstract":"<p>Genetic resistance is the most economical and sustainable approach for crop protection, however, it is regularly overcome by pathogen virulence evolution. Polygenic resistance has greater durability, but unlinked genes are laborious to maintain in breeding programs. Introducing cloned resistance genes into the genome as gene stacks enables polygenic resistance with single locus inheritance. Fielder and Robin wheat plants were generated carrying two loci that each encode five wheat stem rust resistance transgenes, that is, <i>Sr13c</i>/<i>Sr21</i>/<i>Sr22</i>/<i>Sr26</i>/<i>Sr33</i> and <i>Sr22</i>/<i>Sr35</i>/<i>Sr45</i>/<i>Sr50</i>/<i>Sr55</i>. Multiple transgenic events were combined and tested in the field. These lines, with unprecedented levels of transgenic resistance (i.e., 10 transgenes encoded on 90 kb of sequence), showed stable gene stack inheritance and transgene expression after eight generations and were highly resistant in the field. Importantly, in the absence of disease pressure no reproducible differences in the agronomic performance of transgenic lines that contained either one or both gene stacks was seen compared with control lines over two field trial seasons. These data confirm resistance gene stack efficacy and viability as a novel durable resistance strategy in agricultural crop production. Furthermore, this isogenic material that differs only by polygenic resistance provides significant insight into the broader question of the cost of disease resistance in host plants.</p>","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"24 9","pages":"5185-5200"},"PeriodicalIF":12.8,"publicationDate":"2026-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13399084/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148136210","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Correction to “Overexpression of FBR41 Enhances Resistance to Sphinganine Analog Mycotoxin-Induced Cell Death and Alternaria Stem Canker in Tomato”","authors":"","doi":"10.1111/pbi.70721","DOIUrl":"10.1111/pbi.70721","url":null,"abstract":"<p>Shao, Z., Y. Zhao, L. Liu, et al. 2020. “Overexpression of FBR41 Enhances Resistance to Sphinganine Analog Mycotoxin-Induced Cell Death and Alternaria Stem Canker in Tomato.” <i>Journal of Plant Biotechnology</i> 18: 141–154. https://doi.org/10.1111/pbi.13182.</p><p>In the above article, the authors would like to correct the Acknowledgements from ‘This work was supported by the Ministry of Agriculture of China (2016ZX08009003-001), National Natural Science Foundation of China (31200230, 31601746) and Zhejiang Provincial Natural Science Foundation of China (LZ15C150001).’ to ‘This work was supported by the Ministry of Agriculture of China (2016ZX08009003-001), National Natural Science Foundation of China (31830078, 31200230, 31601746) and Zhejiang Provincial Natural Science Foundation of China (LZ15C150001).’</p><p>We apologise for this error.</p>","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"24 9","pages":""},"PeriodicalIF":12.8,"publicationDate":"2026-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/pbi.70721","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148459810","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"KAS-Seq Captures Global Transcription Dynamics and Active Single-Stranded Enhancers in Rice","authors":"Haoxuan Li, Xuling Li, Kayla He, Hua Dang, Wei Shen, Pingxian Zhang, Chuan He, Guanqun Wang","doi":"10.1111/pbi.70677","DOIUrl":"10.1111/pbi.70677","url":null,"abstract":"<p>Transposase-accessible chromatin sequencing (ATAC-seq) has been effectively utilised to generate large-scale open chromatin maps in plants (Wang et al. <span>2020</span>; Li et al. <span>2025</span>). However, ATAC-seq does not precisely indicate active transcriptional activity. In contrast, Ketoxal-Assisted Single-stranded DNA sequencing (KAS-seq) has been widely deployed in capturing genome-wide single-stranded DNA (ssDNA) using N<sub>3</sub>-kethoxal–assisted labelling in situ (Lyu et al. <span>2022</span>), which facilitates fast and accurate analysis of transcription dynamics in mammals (Wu et al. <span>2020</span>). Active chromatin state beyond promoters has been widely applied to predict enhancers in plants, while an active chromatin environment does not necessarily mean an active enhancer (Tippens et al. <span>2020</span>). Since, Pol II is known to bind at certain enhancers (Kim et al. <span>2010</span>), while KAS-seq maps a portion of possible enhancers containing Pol II–mediated transcription bubbles in ssDNA structures and provide a precise foot printing of transcriptional activity (Wu et al. <span>2020</span>). Pol II–engaged enhancers, therefore, can be readily mapped through measuring KAS-seq as well. ATAC-seq serves to identify open chromatin regions that are devoid of nucleosomes, thereby rendering both promoter and enhancer regions accessible for TF binding. However, ATAC-seq signals do not reflect the activity of Pol II elongation, thus failing to identify the active enhancers and their activity. Therefore, the integration of ATAC-seq and KAS-seq probably provides a more accurate prediction of active single-stranded enhancers.</p><p>We therefore deployed the rice seedling photomorphogenesis system (Li et al. <span>2025</span>) to explore application of KAS-seq techniques in rice. At first, we optimised the KAS-seq method using as little as 200 ng gDNA (low input) within rice nuclei (Figure 1a). The dot blot assay showed high compatibility of N<sub>3</sub>-kethoxal–assisted labelling in rice nuclei (Figure 1b). The remaining labelled nuclei were collected for the KAS-seq experiment. We then assigned KAS-seq peaks to gene structure and found the peak distribution was quite different from that of ATAC-seq. Specifically, KAS-seq peaks in the promoter region dramatically reduced to 25% compared with ATAC-seq peaks, which was accompanied by a sharp increase of the peaks assigned to gene body regions (Figure 1c) consistent with the result observed in KAS-seq data derived from human cell (Wu et al. <span>2020</span>). The metagene profile of KAS-seq reads showed a pronounced and sharp peak near the transcription start site (TSS) similar to that of ATAC-seq signals, while a strong, broad peak surrounding the transcription end site (TES) was observed in the KAS-seq reads opposite to the lowest peak intensity at TES in the ATAC-seq reads (Figure 1d). We further observed that the integration of KAS-seq and RNA-seq demonstrated the str","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"24 9","pages":"5010-5012"},"PeriodicalIF":12.8,"publicationDate":"2026-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13399254/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147924970","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"GmDOF3.1-GmCPX Module Regulates Nodulation and Nitrogen Fixation Abilities in Soybean","authors":"Xinzhu Xing, Zhanwu Yang, Zhenqi Shao, Hui Du, Hua Zhang, Huantao Zhang, Xiaobo Huo, Wenlong Li, Youbin Kong, Xihuan Li, Caiying Zhang","doi":"10.1111/pbi.70681","DOIUrl":"10.1111/pbi.70681","url":null,"abstract":"<p>Soybean nodule nitrogen fixation is very important, which can provide a large amount of nitrogen supply for its own growth and that of other crops, but the mechanism is largely unclear. In the present study, a coproporphyrinogen oxidase gene, <i>GmCPX</i>, was identified to facilitate soybean nodulation and nitrogen-fixation under the regulation of transcription factor GmDOF3.1. <i>GmCPX</i> was predominantly expressed in nodule which was highly induced after the inoculation of <i>rhizobium</i>. Overexpression of <i>GmCPX</i> significantly increased nodule numbers, fresh weights, nitrogenase activities, total nitrogen and ammonium nitrogen contents in transgenic soybeans, and also significantly increased the infection cell numbers and areas, heme contents and haemoglobin contents, while significantly decreased the ROS contents. Oppositely, the decreased corresponding characteristics and increased ROS contents were observed in RNAi transgenic soybeans and EMS mutant. The allelic variation analysis of <i>GmCPX</i> in 547 resequencing accessions found that three upstream SNPs were associated with the related traits of nodulation and nitrogen fixation, which further induced a copy number variation of recognition element for transcription factor GmDOF3.1. Further analyses found that GmDOF3.1 negatively regulated the expression of <i>GmCPX</i>, and the opposite phenomena to <i>GmCPX</i> transgenic lines were found in <i>GmDOF3.1</i> overexpression and RNAi transgenic soybeans. Moreover, the genetic variation analysis of <i>GmDOF3.1</i> in resequencing soybeans demonstrated its negative regulation for nodulation and nitrogen fixation. Thus, the regulation of GmDOF3.1-<i>GmCPX</i> module to soybean nodulation and nitrogen fixation was fully verified via forward- and reverse-genetics strategies and could be applied in the genetic improvements of corresponding characteristics in soybean.</p>","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"24 9","pages":"5041-5058"},"PeriodicalIF":12.8,"publicationDate":"2026-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13399125/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147939603","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Cell-Specific Expression and Cellular Compartmental Regulation in Camptothecin Biosynthesis","authors":"Xiaolong Hao, Yinkai Yang, Tiantian Chen, Xiaoxuan Fan, Yiqing Peng, Qingyan Ruan, Qin Zhou, Fanghao Liu, Jiayi He, Yongpeng Li, Yue Feng, Jiyan Qi, Guoyin Kai","doi":"10.1111/pbi.70687","DOIUrl":"10.1111/pbi.70687","url":null,"abstract":"<p>Plant secondary metabolites such as monoterpenoid indole alkaloids (MIAs) show tightly regulated biosynthesis and accumulation in specific organelles of distinct cell types. However, the cell-specific expression patterns of anticancer MIA camptothecin biosynthetic genes in <i>Ophiorrhiza pumila</i> and the regulatory mechanisms of camptothecin biosynthesis via cellular compartmentalisation remain poorly understood. Here, single-cell RNA sequencing of <i>O. pumila</i> leaves generated 9181 cells, classified into four types using marker gene annotation and in situ hybridisation. Gene expression profiling combined with pseudotime analyses revealed that camptothecin biosynthetic genes are preferentially expressed in epidermal and young cells. Notably, the genes encoding camptothecin key enzymes, strictosidine synthase (STR) and tryptophan decarboxylase (TDC), have expanded within the <i>O. pumila</i> <i>STR-TDC</i> gene cluster, supporting cell-specific specialisation. Concurrently, this gene cluster has evolved a novel functional gene, <i>OpAVT1</i>, which annotated as a vacuolar transporter, mediates the transport of tryptophan from vacuole to the cytosol, thereby regulating camptothecin and other MIAs accumulation. Collectively, these findings establish a cellular-level framework for camptothecin biosynthesis and regulation.</p>","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"24 9","pages":"5114-5130"},"PeriodicalIF":12.8,"publicationDate":"2026-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398565/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148025400","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Haifan Wen, Hui Du, Siqi Wang, Yiqun Weng, Juan Liu, Huanle He, Run Cai, Jian Pan, Junsong Pan
{"title":"A Novel AP2/ERF Transcription Factor Controls Trichome Initiation and Fruit Development in Cucumber","authors":"Haifan Wen, Hui Du, Siqi Wang, Yiqun Weng, Juan Liu, Huanle He, Run Cai, Jian Pan, Junsong Pan","doi":"10.1111/pbi.70676","DOIUrl":"10.1111/pbi.70676","url":null,"abstract":"<p>Trichomes, the hair-like outgrowths of the plant epidermis, are critical for defence, reproduction and development. However, the underlying molecular mechanisms governing their initiation remain elusive. Here, we identified CsGL2, an AP2/ERF transcription factor, as a key integrator controlling trichome initiation and fruit development in cucumber. Genetic and molecular studies indicate that CsGL2 activates <i>CsGL1</i>/<i>Mict</i>, subsequently triggering the morphogenesis of multicellular trichomes. Beyond epidermal fate, CsGL2 upregulates the tubercle regulator <i>Tu</i> together with the cytokinin rate-limiting enzyme gene <i>IPT1</i>, thereby initiating fruit tubercles through cytokinin-linked pathways. Notably, we found that ovule-specific CsGL2 activates the parthenocarpy gene <i>CsNPF1</i>, and <i>CsGL2</i> overexpression markedly increases the frequency of seedless fruit set. Thus, our findings establish a switch-based mechanism for progressive cell-fate specification in multicellular epidermal organs. This reveals how a single transcription factor integrates transcriptional and hormonal pathways to coordinate distinct developmental programmes.</p>","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"24 9","pages":"5077-5093"},"PeriodicalIF":12.8,"publicationDate":"2026-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13399041/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147969153","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Aye Aye Htun, Ya Wang, Jiahao Liu, Zhiqiang Guo, Yi Zheng, Wenjie Wang, Shuting Ma, Aolin Jia, Yan Ren, Feng Chen
{"title":"MYB Factors: Hubs of Plant Stress and Hormone Crosstalk","authors":"Aye Aye Htun, Ya Wang, Jiahao Liu, Zhiqiang Guo, Yi Zheng, Wenjie Wang, Shuting Ma, Aolin Jia, Yan Ren, Feng Chen","doi":"10.1111/pbi.70694","DOIUrl":"10.1111/pbi.70694","url":null,"abstract":"<p>MYB transcription factors function as main regulatory hubs that integrate environmental signals with multi-hormonal pathways to synchronize plant growth, metabolism and stress responses. This review delineates the regulatory roles of MYB in the signalling pathway of salicylic acid, jasmonic acid, ethylene and abscisic acid signalling. These are responsible for regulating hormone biosynthesis and facilitating interaction between biotic and abiotic stress responses. The post-translational regulation of MYB activity by kinase cascades and the direct regulation of secondary metabolic pathways, such as the biosynthesis of phenylpropanol and lignin, by MYBs through the MYB-bHLH-WD40 (MBW) complex were investigated. MYB transcription factors located at the intersection of hormone signalling, metabolic regulation, and developmental regulation play a unique role in coordinating plant adaptation from the cellular level to the whole plant.</p>","PeriodicalId":221,"journal":{"name":"Plant Biotechnology Journal","volume":"24 9","pages":"5201-5223"},"PeriodicalIF":12.8,"publicationDate":"2026-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13399108/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148136147","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}