Candidate genes associated with low temperature tolerance in cucumber adult plants identified by combining GWAS & QTL mapping

Abstract

Fruit quality and yield are reduced when cucumber (Cucumis sativus L.) plants are exposed to low temperature (LT) stress, yet, the inheritance and genes linked to cold tolerance in adult plants have not been reported yet. Here, the LT-tolerance of 120 cucumber accessions representing four ecotypes were evaluated by GWAS, and also, in 140 recombinant inbred lines (RILs) derived from a biparental cross. Plants were exposed to naturally occurring LT environments in a plastic greenhouse, in winter 2022, and 2023, and a low temperature injury index (LTII) was employed to evaluate plant performance. Genetic analysis revealed that the LT-tolerance evaluated in the adult cucumber plants was a multigenic quantitative trait, and that 18 of the 120 accessions were highly LT tolerant by our LTII assessment. Two loci (gLTT1.1 and gLTT3.1) exhibited strong signals that were consistent and stable in two environments. In addition, two QTLs—qLTT1.2 on chromosome (Chr.) 1, and qLTT3.1 on Chr. 3, were discovered in all tests using RIL population derived from a cross between LT-sensitive ‘CsIVF0106’, and LT-tolerant ‘CsIVF0168’. qLTT1.2 was delimited to a 1.24-Mb region and qLTT3.1 was narrowed to a 1.43-Mb region. Interestingly, a peak single nucleotide polymorphism (SNP) at gLTT1.1 and gLTT3.1 was also found in qLTT1.2 and qLTT3.1, respectively. These loci were thus renamed as gLTT1.1 and gLTT3.1. In these regions, 25 genes were associated with the LT response. By identifying differences in haplotypes and transcript profiles among these genes, we identified four candidates: CsaV3_1G012520 (an ethylene-responsive transcription factor) and CsaV3_1G013060 (a RING/U-box superfamily protein) in gLTT1.1, and two RING-type E3 ubiquitin transferases at CsaV3_3G018440 and CsaV3_3G017700 in gLTT3.1 that may regulate LT-tolerance in adult cucumber. Interestingly, the accessions in which the LT-tolerant haplotypes for two loci were pyramided, displayed maximally high tolerance for LT. These findings therefore provide a solid foundation for the identification of LT-tolerant genes and the molecular breeding of cucumber with LT-tolerance.

References

[] An JP, Wang XF, Zhang XW, Xu HF, Bi SQ, You CX, Hao YJ. An apple MYB transcription factor regulates cold tolerance and anthocyanin accumulation and undergoes MIEL1-mediated degradation. Plant Biotechnol J, 2020, 18(2): 337-353

[] Bai C, Fang M, Zhai B, Ma L, Fu A, Gao L, Kou X, Meng D, Wang Q, Zheng S. Regulations of m6A methylation on tomato fruit chilling injury. Horticultural Plant J, 2021, 7(5): 434-442

[] Byun MY, Cui LH, Oh TK, Jung Y-J, Lee A, Park KY, Kang BG, Kim WT (2017) Homologous U-box E3 ubiquitin ligases OsPUB2 and OsPUB3 are involved in the positive regulation of low temperature stress response in rice (Oryza sativa L.). Front Plant Sci 8:16. https://doi.org/10.3389/fpls.2017.00016

[] Cabrera RM, Saltveit ME, Owens K (1992) Cucumber cultivars differ in their response to chilling temperatures. J Am Soc Hortic Sci 117 (5):802–807. https://doi.org/10.21273/JASHS.117.5.802

[] Chen J, Li Y, Li F, Wu Q, Jiang Y, Yuan D. Banana MaABI5 is involved in ABA-induced cold tolerance through interaction with a RING E3 ubiquitin ligase, MaC3HC4-1. Sci Hortic, 2018, 237: 239-246

[] Chung SM, Staub JE, Fazio G (2003) Inheritance of chilling injury: a maternally inherited trait in cucumber. J Am Soc Hortic Sci 128 (4):526–530. https://doi.org/10.21273/JASHS.128.4.0526

[] Diao P, Chen C, Zhang Y, Meng Q, Lv W, Ma N. The role of NAC transcription factor in plant cold response. Plant Signal Behav, 2020, 15(9): 1785668

[] Ding Y, Shi Y, Yang S. Advances and challenges in uncovering cold tolerance regulatory mechanisms in plants. New Phytol, 2019, 222(4): 1690-1704

[] Dong CH, Agarwal M, Zhang Y, Xie Q, Zhu JK. The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1. Proc Natl Acad Sci, 2006, 103(21): 8281-8286

[] Dong S, Wang W, Bo K, Miao H, Song Z, Wei S, Zhang S, Gu X. Quantitative Trait Loci Mapping and Candidate Gene Analysis of Low Temperature Tolerance in Cucumber Seedlings. Front Plant Sci, 2019, 10: 1620

[] Dong S, Li C, Tian H, Wang W, Yang X, Beckles DM, Liu X, Guan J, Gu X, Sun J. Natural variation in STAYGREEN contributes to low temperature tolerance in cucumber. J Integr Plant Biol, 2023, 65(12): 2552-2568

[] Dong H (2017) Mining genes with cold tolerance of elite germplasm in cucumber. [Doctoral dissertation]. Chinese Academy Agriculture Science, Beijing

[] Fang H, Meng Q, Xu J, Tang H, Tang S, Zhang H, Huang J. Knock-down of stress inducible OsSRFP1 encoding an E3 ubiquitin ligase with transcriptional activation activity confers abiotic stress tolerance through enhancing antioxidant protection in rice. Plant Mol Biol, 2015, 87: 441-458

[] Haas BJ, Delcher AL, Mount SM, Wortman JR, Smith Jr RK, Hannick LI, Maiti R, Ronning CM, Rusch DB, Town CD (2003) Improving the Arabidopsis genome annotation using maximal transcript alignment assemblies. Nucleic Acids Res 31(19):5654–5666. https://doi.org/10.1093/nar/gkg770

[] Huang J, Zhao X, Bürger M, Chory J, Wang X. The role of ethylene in plant temperature stress response. Trends Plant Sci, 2023, 28(7): 808-824

[] Jiang H, Shi Y, Liu J, Li Z, Fu D, Wu S, Li M, Yang Z, Shi Y, Lai J, Yang X, Gong Z, Hua J, Yang S. Natural polymorphism of ZmICE1 contributes to amino acid metabolism that impacts cold tolerance in maize. Nat Plants, 2022, 8(10): 1176-1190

[] Kidokoro S, Shinozaki K, Yamaguchi-Shinozaki K (2022) Transcriptional regulatory network of plant cold-stress responses. Trends Plant Sci 27 (9):922–935 https://doi.org/10.1016/j.tplants.2022.01.008

[] Klay I, Gouia S, Liu M, Mila I, Khoudi H, Bernadac A, Bouzayen M, Pirrello J. Ethylene Response Factors (ERF) are differentially regulated by different abiotic stress types in tomato plants. Plant Sci, 2018, 274: 137-145

[] Kłosińska U, Kozik EU, Nowicki M, Wehner TC. Low temperature seed germination of cucumber: genetic basis of the tolerance trait. J Horticultural Res, 2013, 21(2): 125-130

[] Kozik E, Klosinska U, Wehner T (2012) Inheritance of low temperature seed germination ability in cucumber. In: Cucurbitaceae 2012. Proceedings of the Xth EUCARPIA Meeting on Genetics and Breeding of Cucurbitaceae, Antalya, Turkey, 15–18 October, 2012, 2012. University of Cukurova, Ziraat Fakultesi, pp 575–578

[] Kozik EU, Wehner TC. A single dominant gene Ch for chilling resistance in cucumber seedlings. J Am Soc Hortic Sci, 2008, 133(2): 225-227

[] Lee H, Xiong L, Gong Z, Ishitani M, Stevenson B, Zhu JK. The Arabidopsis HOS1 gene negatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo–cytoplasmic partitioning. Genes Dev, 2001, 15(7): 912-924

[] Li B, Wang X, Wang X, Xi Z. An AP2/ERF transcription factor VvERF63 positively regulates cold tolerance in Arabidopsis and grape leaves. Environ Exp Bot, 2023, 205 105124

[] Li H, Zhu W, Peng J. Inheritance analysis and screening of linked marker of chilling resistance in cucumber (Cucumis sativus L.). J Shanghai Jiaotong University (Agricultural Science), 2015, 33(1): 14-18

[] Li H, Ding Y, Shi Y, Zhang X, Zhang S, Gong Z, Yang S (2017) MPK3-and MPK6-mediated ICE1 phosphorylation negatively regulates ICE1 stability and freezing tolerance in Arabidopsis. Dev Cell 43 (5):630–642. e634. https://doi.org/10.1016/j.devcel.2017.09.025

[] Li C, Dong S, Beckles DM, Liu X, Liu D, Miao H, Zhang S, Gu X (2022a) Identification of QTLs controlling cold tolerance in cucumber (Cucumis sativus L.) seedlings. Scientia Horticulturae 305. https://doi.org/10.3390/plants10010085

[] Li C, Dong S, Beckles DM, Miao H, Sun J, Liu X, Wang W, Zhang S, Gu X (2022b) The qLTG1.1 candidate gene CsGAI regulates low temperature seed germination in cucumber. Theor Appl Genet. https://doi.org/10.1007/s00122-022-04097-w

[] Li C, Dong S, Beckles DM, Liu X, Guan J, Gu X, Miao H, Zhang S (2023b) GWAS reveals novel loci and identifies a pentatricopeptide repeat-containing protein (CsPPR) that improves low temperature germination in cucumber. Front Plant Sci 14. https://doi.org/10.3389/fpls.2023.1116214

[] Liu Y, Khan AR, Gan Y. C2H2 zinc finger proteins response to abiotic stress in plants. Int J Mol Sci, 2022, 23(5): 2730

[] Liu W, Chen H, Jin Y, Qi H. Advances in alcohol dehydrogenase enzymes and their gene families in higher plants. Plant Physiol J, 2014, 50(10): 1479-1493

[] Liu D, Dong S, Miao H, Liu X, Li C, Han J, Zhang S, Gu X (2022a) A Large-Scale Genomic Association Analysis Identifies the Candidate Genes Regulating Salt Tolerance in Cucumber (Cucumis sativus L.) Seedlings. Int J Mol Sci 23(15) . https://doi.org/10.1111/pbi.13452

[] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods, 2001, 25(4): 402-408

[] Lourenço T, Sapeta H, Figueiredo DD, Rodrigues M, Cordeiro A, Abreu IA, Saibo NJ, Oliveira MM. Isolation and characterization of rice (Oryza sativa L.) E3-ubiquitin ligase OsHOS1 gene in the modulation of cold stress response. Plant Mol Biol, 2013, 83: 351-363

[] Meng L, Li H, Zhang L, Wang J. QTL IciMapping: integrated software for genetic linkage map construction and quantitative trait locus mapping in biparental populations. Crop J, 2015, 3(3): 269-283

[] Miao Y, Ning Y, Cao Y, Shen J, Pang X, Cui L, Cheng C, Chen J. Evaluation of cucumber's chilling tolerance at germination and seedling stages. J Applied Ecol, 2013, 24(7): 1914-1922

[] Mittler R, Zandalinas SI, Fichman Y, Van Breusegem F. Reactive oxygen species signalling in plant stress responses. Nat Rev Mol Cell Biol, 2022, 23(10): 663-679

[] Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2012) AP2/ERF family transcription factors in plant abiotic stress responses. Biochim Biophys Acta-Gene Regul Mech 1819 (2):86–96. https://doi.org/10.1016/j.bbagrm.2011.08.004

[] Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, De Bakker PI, Daly MJ. PLINK: a tool set for whole-genome association and population-based linkage analyses. The American Journal of Human Genetics, 2007, 81(3): 559-575

[] Qi X, Zhang P, Xu Q, Chen X. Evaluation of cold tolerance of cucumber seed and seedling. China Vegetables, 2011, 16: 34-38

[] Qi C, Dong D, Li Y, Wang X, Guo L, Liu L, Dong X, Li X, Yuan X, Ren S, Zhang N, Guo YD. Heat shock-induced cold acclimation in cucumber through CsHSFA1d-activated JA biosynthesis and signaling. Plant J, 2022, 111(1): 85-102

[] Qi J, Liu X, Shen D, Miao H, Xie B, Li X, Zeng P, Wang S, Shang Y, Gu X (2013) A genomic variation map provides insights into the genetic basis of cucumber domestication and diversity. Nature genet 45 (12):1510–1515. https://doi.org/10.1038/ng.2801

[] Shi L, Yang Y, Xie Q, Miao H, Bo K, Song Z, Wang Y, Xie B, Zhang S, Gu X (2018) Inheritance and QTL mapping of cucumber mosaic virus resistance in cucumber (Cucumis Sativus L.). PLoS One 13 (7):e0200571. https://doi.org/10.1371/journal.pone.0200571

[] Smeets L, Wehner TC. Environmental effects on genetic variation of chilling resistance in cucumber. Euphytica, 1997, 97(2): 217-225

[] Song L, Tan Z, Zhang W, Li Q, Jiang Z, Shen S, Luo S, Chen X. Exogenous melatonin improves the chilling tolerance and preharvest fruit shelf life in eggplant by affecting ROS-and senescence-related processes. Horticultural Plant Journal, 2023, 9(3): 523-540

[] Song Z, Wang W, Shi L, Zhang S, Xie Q, Wei S, Wang Y, Bo K, Miao H, Zhang S, Gu X, Havey M (2018) Identification of QTLs controlling low-temperature tolerance during the germination stage in cucumber (Cucumis sativus L.). Plant Breed 137 (4):629–637. https://doi.org/10.1111/pbr.12601

[] Sun X, Zhao T, Gan S, Ren X, Fang L, Karungo SK, Wang Y, Chen L, Li S, Xin H. Ethylene positively regulates cold tolerance in grapevine by modulating the expression of ETHYLENE RESPONSE FACTOR 057. Sci Rep, 2016, 6(1): 24066

[] Tang Y, Chen H, Deng T, Chang Y, Sun K, Ditta A, Khan MKR, Wang K, Wang B. Genome-wide identification and analysis of the GUB_WAK_bind gene family in Gossypium hirsutum. Mol Biol Rep, 2022, 49(7): 6405-6413

[] Tian C, Zhai L, Zhu W, Qi X, Yu Z, Wang H, Chen F, Wang L, Chen S. Characterization of the TCP Gene Family in Chrysanthemum nankingense and the Role of CnTCP4 in Cold Tolerance. Plants, 2022, 11(7): 936

[] Turner SD (2014) qqman: an R package for visualizing GWAS results using QQ and manhattan plots. J Open Sour Softw 3(25):731. https://doi.org/10.21105/joss.00731

[] Wan H, Zhao Z, Qian C, Sui Y, Malik AA, Chen J. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem, 2010, 399(2): 257-261

[] Wang K, Ding Y, Cai C, Chen Z, Zhu C. The role of C2H2 zinc finger proteins in plant responses to abiotic stresses. Physiol Plant, 2019, 165(4): 690-700

[] Wang W, Song Z, Bo K, Dong S, Wei S, Miao H, Li J, Zhang S, Gu X. Evaluation and genome-wide association study (GWAS) of low-temperature tolerance at seedling stage in cucumber core germplasm. J Plant Genet Resour, 2019, 20(6): 1606-1612

[] Wang Y, Jiang H, Mao Z, Liu W, Jiang S, Xu H, Su M, Zhang J, Wang N, Zhang Z. Ethylene increases the cold tolerance of apple via the MdERF1B–MdCIbHLH1 regulatory module. Plant J, 2021, 106(2): 379-393

[] Wang S, Lv X, Zhang J, Chen D, Chen S, Fan G, Ma C, Wang Y. Roles of E3 ubiquitin ligases in plant responses to abiotic stresses. Int J Mol Sci, 2022, 23(4): 2308

[] Wang P, Liu WC, Han C, Wang S, Bai MY, Song CP. Reactive Oxygen Species: Multidimensional Regulators of Plant Adaptation to Abiotic Stress and Development. J Integr Plant Biol, 2023

[] Wang X, Ding Y, Li Z, Shi Y, Wang J, Hua J, Gong Z, Zhou J-M, Yang S (2019c) PUB25 and PUB26 promote plant freezing tolerance by degrading the cold signaling negative regulator MYB15. Dev cell 51 (2):222–235. https://doi.org/10.1016/j.devcel.2019.08.008

[] Wang Y, Cao S, Sui X, Wang J, Geng Y, Gao F, Zhou Y (2022b) Genome-wide characterization, evolution, and expression analysis of the ascorbate peroxidase and glutathione peroxidase gene families in response to cold and osmotic stress in Ammopiptanthus nanus. J Plant Growth Regul:1–21. https://doi.org/10.1007/s00344-021-10570-5

[] Wang Z, Dong S, Liu Y, Beckles DM, Li C, Han J, Zhang Y, Liu X, Guan J, Gu X (2023b) A Genome-wide association study identifies candidate genes for heat tolerance in adult cucumber (Cucumis sativus L.) plants. Hortic Plant J. https://doi.org/10.1016/j.hpj.2023.11.001

[] Wang H. Evaluation of low temperature tolerance and QTL mapping in cucumber germplasm [Doctoral dissertation], 2014 Beijing Chinese Academy of Agriculture Science

[] Wei S, Zhang S, Bo K, Wang W, Miao H, Dong S, Gu X, Zhang S (2019). Evaluation and genome-wide association study (GWAS) of seedling thermotolerance in cucumber core germplasm. J Plant Genet Res 20:1223-1231. https://doi.org/10.13430/j.cnki.jpgr.20181226001

[] Woffelman C. DNAMAN for Windows. Lynon Biosoft, Institute of Molecular Plant Sciences, Leiden University, Leiden, 2004, 5: 10

[] Wu Y, Wu S, Wang X, Mao T, Bao M, Zhang J, Zhang J. Genome-wide identification and characterization of the bHLH gene family in an ornamental woody plant Prunus mume. Horticultural Plant Journal, 2022, 8(4): 531-544

[] Xie Y, Chen P, Yan Y, Bao C, Li X, Wang L, Shen X, Li H, Liu X, Niu C. An atypical R2R3 MYB transcription factor increases cold hardiness by CBF-dependent and CBF-independent pathways in apple. New Phytol, 2018, 218(1): 201-218

[] Xu W, Zhang N, Jiao Y, Li R, Xiao D, Wang Z. The grapevine basic helix-loop-helix (bHLH) transcription factor positively modulates CBF-pathway and confers tolerance to cold-stress in Arabidopsis. Mol Biol Rep, 2014, 41: 5329-5342

[] Xu G, Li L, Zhou J, Lyu D, Zhao D, Qin S. Comparison of transcriptome and metabolome analysis revealed differences in cold resistant metabolic pathways in different apple cultivars under low temperature stress. Horticultural Plant Journal, 2023, 9(2): 183-198

[] Yagcioglu M, Jiang B, Wang P, Wang Y, Ellialtioglu SS, Weng Y. QTL mapping of low temperature germination ability in cucumber. Euphytica, 2019, 215(4): 1-11

[] Yan Y, Mintao S, Si M, Qian F, Yijia W, Qinghua D, Mengdi Z, Chaoxing H, Yansu L, Lihong G, Xianchang Y (2022) Mechanism of CsGPA1 in regulating cold tolerance of cucumber. Hortic Res 9:uhac109. https://doi.org/10.1093/hr/uhac109

[] Yang T, Chaudhuri S, Yang L, Du L, Poovaiah B. A calcium/calmodulin-regulated member of the receptor-like kinase family confers cold tolerance in plants. J Biol Chem, 2010, 285(10): 7119-7126

[] Yang J, Guo X, Mei Q, Qiu L, Chen P, Li W, Mao K, Ma F. MdbHLH4 negatively regulates apple cold tolerance by inhibiting MdCBF1/3 expression and promoting MdCAX3L-2 expression. Plant Physiol, 2023, 191(1): 789-806

[] Yu JQ, Zhou YH, Ye SF, Huang LF. 24-epibrassinolide and abscisic acid protect cucumber seedlings from chilling injury. J Hortic Sci Biotechnol, 2002, 77(4): 470-473

[] Zhang Y, Yu H, Yang X, Li Q, Ling J, Wang H, Gu X, Huang S, Jiang W. CsWRKY46, a WRKY transcription factor from cucumber, confers cold resistance in transgenic-plant by regulating a set of cold-stress responsive genes in an ABA-dependent manner. Plant Physiol Biochem, 2016, 108: 478-487

[] Zhang H, Guo J, Chen X, Zhou Y, Pei Y, Chen L, ul Haq S, Zhang M, Gong H, Chen R. Transcription factor CabHLH035 promotes cold resistance and homeostasis of reactive oxygen species in pepper. Horticultural Plant Journal, 2023

[] Zhang Z, Li J, Li F, Liu H, Yang W, Chong K, Xu Y (2017) OsMAPK3 phosphorylates OsbHLH002/OsICE1 and inhibits its ubiquitination to activate OsTPP1 and enhances rice chilling tolerance. Dev Cell 43 (6):731–743. https://doi.org/10.1016/j.devcel.2017.11.016

[] Zhang L, Jiang X, Liu Q, Ahammed GJ, Lin R, Wang L, Shao S, Yu J, Zhou Y. The HY5 and MYB15 transcription factors positively regulate cold tolerance in tomato via the CBF pathway. Plant Cell Environ, 2020, 43(11): 2712-2726

[] Zhang Y, Ming R, Khan M, Wang Y, Dahro B, Xiao W, Li C, Liu JH (2022) ERF9 of Poncirus trifoliata (L.) Raf. undergoes feedback regulation by ethylene and modulates cold tolerance via regulating a glutathione S‐transferase U17 gene. Plant Biotechnol J 20(1):183–200. https://doi.org/10.1111/pbi.13705

[] Zhao C, Wang P, Si T, Hsu C, Wang L, Zayed O, Yu Z, Zhu Y, Dong J, Tao WA (2017) MAP kinase cascades regulate the cold response by modulating ICE1 protein stability. Dev Cell 43 (5):618–629. e615. https://doi.org/10.3389/fpls.2016.01599

[] Zhou M, Li W, Zheng Y, Lin P, Yao X, Lin J. CbRCI35, a cold responsive peroxidase from Capsella bursa-pastoris regulates reactive oxygen species homeostasis and enhances cold tolerance in tobacco. Front Plant Sci, 2016, 7: 1599

[] Zhuo X, Zheng T, Zhang Z, Zhang Y, Jiang L, Ahmad S, Sun L, Wang J, Cheng T, Zhang Q. Genome-wide analysis of the NAC transcription factor gene family reveals differential expression patterns and cold-stress responses in the woody plant Prunus mume. Genes, 2018, 9(10): 494

Funding

National Natural Science Foundation of China(No.31902028)