Selection and Validation of Reference Genes for RT-qPCR in Allium tuberosum Infected by Botrytis squamosa

doi:10.13560/j.cnki.biotech.bull.1985.2022-0851

Abstract

Abstract:

Chinese chive gray mold caused by Botrytis squamosa is one of the major factors affecting the production and quality of Chinese chive（Allium tuberosum）. In order to screen stable reference genes in Chinese chive leaves infected with gray mold for quantitative gene expression analysis, Chinese chive leaves mock-inoculated and inoculated with B. squamosa for 24, 48 and 72 h were taken as materials. Thirteen genes including UBC1, UBC2, UBQ1, UBQ2, GAPDH3, GAPDH4, TUB, EF-1α, 40S RP, DDX, eIF-1A, PABP and DnaJ were selected as candidate reference genes based on previous RNA-Seq data of Chinese chive. The expressions of the candidate reference genes were detected by real-time quantitative PCR（RT-qPCR）and evaluated by geNorm, NormFinder, BestKeeper and RefFinder statistical algorithms. The results showed that among the thirteen candidate reference genes, UBQ1 had the smallest variation range of Ct value and the most stable expression. The most suitable reference genes evaluated by geNorm, NormFinder and BestKeeper software were different. Comprehensive evaluation of RefFinder website showed that UBC2 and UBQ1 had the better stability in Chinese chive leaves after inoculation with B. squamosa, and DDX displayed the lower stability. In order to verify the reliability of the selected reference genes, six candidate genes with different stability were used as internal controls for quantitative analysis, and normalized the expression levels of GST676 and PRP902 in Chinese chive leaves at different time points after inoculation with B. squamosa. The expression trends of GST676 and PRP902 corrected by UBC2 or UBQ1 with fine stability were consistent with that in RNA-Seq data, while the expression trends of GST676 and PRP902 corrected by 40S RP, eIF-1A, GAPDH4 or DDX with relative poor stability had some difference with that in RNA-Seq data. These results of software analysis and experimental verification showed that UBC2 and UBQ1 were the most suitable reference genes in Chinese chive leaves infected by B. squamosa for quantitative expression analysis. The results lay the foundation for future research on the expression and function of key genes in Chinese chives leaves during infection with B. squamosa.

Key words: Allium tuberosum, Botrytis squamosa, reference gene, real-time quantitative PCR, expression stability, gray mold

SONG Hai-na, WU Xin-tong, YANG Lu-yu, GENG Xi-ning, ZHANG Hua-min, SONG Xiao-long. Selection and Validation of Reference Genes for RT-qPCR in Allium tuberosum Infected by Botrytis squamosa[J]. Biotechnology Bulletin, 2023, 39(3): 101-115.

Figures/Tables 12

References 55

[1]	黄征. 葱蒜类蔬菜真菌病害调查及其病原鉴定[J]. 中国农学通报, 2007, 23(5): 326-329.
	Huang Z. Diagnosis and identification on the fungal disease of bulb crops in Fujian[J]. Chin Agric Sci Bull, 2007, 23(5): 326-329. doi: 10.11924/j.issn.1000-6850.0705326
[2]	崔蕴刚, 张华敏, 李延龙, 等. 韭菜灰霉病病原鉴定及其生物学特性[J]. 北方园艺, 2020(4): 14-19.
	Cui YG, Zhang HM, Li YL, et al. Identification and biological characterization of the pathogen of Allium tuberosum grey mold[J]. North Hortic, 2020(4): 14-19.
[3]	Zhang J, Zhang L, Li GQ, et al. Botrytis sinoallii: a new species of the grey mould pathogen on Allium crops in China[J]. Mycoscience, 2010, 51(6): 421-431. doi: 10.47371/mycosci.MYC51421 URL
[4]	胡彬, 黄中乔, 刘西莉, 等. 9种杀菌剂对韭菜灰霉病的防治效果[J]. 中国农学通报, 2014, 30(4): 293-298.
	Hu B, Huang ZQ, Liu XL, et al. Control efficacy of 9 fungicides against Chinese chives gray mold[J]. Chin Agric Sci Bull, 2014, 30(4): 293-298. doi: 10.11924/j.issn.1000-6850.2013-3021
[5]	胡彬, 李琳, 戚如诗, 等. 从韭菜腐霉利残留超标看农药登记及最大残留限量标准的科学制定[J]. 中国蔬菜, 2020(5): 9-11.
	Hu B, Li L, Qi RS, et al. The scientific establishment of pesticide registration and maximum residue limit standard from the point of Chinese chives procymidone residue exceed the standard[J]. China Veg, 2020(5): 9-11.
[6]	高皓杰, 张兰云, 李桐桐, 等. 韭菜灰霉病防治烟剂的筛选与评价[J]. 农药学学报, 2022, 24(2): 315-325.
	Gao HJ, Zhang LY, Li TT, et al. Screening and evaluation of smoke generators to control gray mold of Chinese chives[J]. Chin J Pestic Sci, 2022, 24(2): 315-325.
[7]	韩之琪, 贲海燕, 谢学文, 等. 灰葡萄孢对杀菌剂抗性研究进展[J]. 中国蔬菜, 2014(5): 6-10.
	Han ZQ, Ben HY, Xie XW, et al. Research progress on Botrytis cinerea resistance to different fungicides[J]. China Veg, 2014(5): 6-10.
[8]	Rupp S, Plesken C, Rumsey S, et al. Botrytis fragariae, a new species causing gray mold on strawberries, shows high frequencies of specific and efflux-based fungicide resistance[J]. Appl Environ Microbiol, 2017, 83(9): e00269-e00217.
[9]	胡适宜. 被子植物胚胎学[M]. 北京: 人民教育出版社, 1982.
	Hu SY. Angiosperm embryology[M]. Beijing: People’s Education Press, 1982.
[10]	张华敏, 陈建华, 尹守恒, 等. 韭菜无融合生殖种子形成机制研究进展[J]. 河南农业科学, 2020, 49(6): 1-7.
	Zhang HM, Chen JH, Yin SH, et al. Research progress of the formation mechanism of apomictic seed in Chinese chive[J]. J Henan Agric Sci, 2020, 49(6): 1-7.
[11]	Gachon C, Mingam A, Charrier B. Real-time PCR: what relevance to plant studies?[J]. J Exp Bot, 2004, 55(402): 1445-1454. doi: 10.1093/jxb/erh181 pmid: 15208338
[12]	Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR[J]. Nat Protoc, 2006, 1(3): 1559-1582. doi: 10.1038/nprot.2006.236 pmid: 17406449
[13]	Dheda K, Huggett JF, Chang JS, et al. The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization[J]. Anal Biochem, 2005, 344(1): 141-143. doi: 10.1016/j.ab.2005.05.022 pmid: 16054107
[14]	袁伟, 万红建, 杨悦俭. 植物实时荧光定量PCR内参基因的特点及选择[J]. 植物学报, 2012, 47(4): 427-436. doi: 10.3724/SP.J.1259.2012.00427
	Yuan W, Wan HJ, Yang YJ. Characterization and selection of reference genes for real-time quantitative RT-PCR of plants[J]. Chin Bull Bot, 2012, 47(4): 427-436.
[15]	Brunner AM, Yakovlev IA, Strauss SH. Validating internal controls for quantitative plant gene expression studies[J]. BMC Plant Biol, 2004, 4: 14. pmid: 15317655
[16]	Huggett J, Dheda K, Bustin S, et al. Real-time RT-PCR normalisation; strategies and considerations[J]. Genes Immun, 2005, 6(4): 279-284. doi: 10.1038/sj.gene.6364190 pmid: 15815687
[17]	Jain M, Nijhawan A, Tyagi AK, et al. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR[J]. Biochem Biophys Res Commun, 2006, 345(2): 646-651. doi: 10.1016/j.bbrc.2006.04.140 URL
[18]	蒋婷婷, 高燕会, 童再康. 石蒜属植物实时荧光定量PCR内参基因的选择[J]. 园艺学报, 2015, 42(6): 1129-1138. doi: 10.16420/j.issn.0513-353x.2014-0999
	Jiang TT, Gao YH, Tong ZK. Selection of reference genes for quantitative real-time PCR in Lycoris[J]. Acta Hortic Sin, 2015, 42(6): 1129-1138.
[19]	马璐琳, 段青, 崔光芬, 等. 钝裂银莲花花色素合成相关基因RT-qPCR内参基因的筛选[J]. 园艺学报, 2021, 48(2): 377-388. doi: 10.16420/j.issn.0513-353x.2020-0306
	Ma LL, Duan Q, Cui GF, et al. Selection and validation of reference genes for RT-qPCR analysis of the correlated genes in flower pigments biosynthesis pathway of Anemone obtusiloba[J]. Acta Hortic Sin, 2021, 48(2): 377-388.
[20]	李梦倩, 刘敏, 张蒙, 等. 大蒜体细胞胚发生mRNA及miRNA qPCR内参基因的筛选和验证[J]. 农业生物技术学报, 2021, 29(12): 2449-2464.
	Li MQ, Liu M, Zhang M, et al. Identification and verification of somatic embryogenesis mRNA and miRNA qPCR reference genes in garlic(Allium sativum)[J]. J Agric Biotechnol, 2021, 29(12): 2449-2464.
[21]	Gutierrez L, Mauriat M, Guénin S, et al. The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction(RT-PCR)analysis in plants[J]. Plant Biotechnol J, 2008, 6(6): 609-618. doi: 10.1111/j.1467-7652.2008.00346.x pmid: 18433420
[22]	Volkov RA, Panchuk II, Schöffl F. Heat-stress-dependency and developmental modulation of gene expression: the potential of house-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCR[J]. J Exp Bot, 2003, 54(391): 2343-2349. pmid: 14504302
[23]	Hu RB, Fan CM, Li HY, et al. Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR[J]. BMC Mol Biol, 2009, 10: 93. doi: 10.1186/1471-2199-10-93 pmid: 19785741
[24]	Die JV, Román B, Nadal S, et al. Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions[J]. Planta, 2010, 232(1): 145-153. doi: 10.1007/s00425-010-1158-1 URL
[25]	Kozera B, Rapacz M. Reference genes in real-time PCR[J]. J Appl Genet, 2013, 54(4): 391-406. pmid: 24078518
[26]	张玉芳, 赵丽娟, 曾幼玲. 基因表达研究中内参基因的选择与应用[J]. 植物生理学报, 2014, 50(8): 1119-1125.
	Zhang YF, Zhao LJ, Zeng YL. Selection and application of reference genes for gene expression studies[J]. Plant Physiol J, 2014, 50(8): 1119-1125.
[27]	Deo RC, Bonanno JB, Sonenberg N, et al. Recognition of polyadenylate RNA by the poly(A)-binding protein[J]. Cell, 1999, 98(6): 835-845. pmid: 10499800
[28]	Rocak S, Linder P. DEAD-box proteins: the driving forces behind RNA metabolism[J]. Nat Rev Mol Cell Biol, 2004, 5(3): 232-241. doi: 10.1038/nrm1335
[29]	蔡敬, 孟小庆, 董婷婷, 等. DEAD-box解旋酶在植物非生物胁迫响应中的功能研究进展[J]. 生命科学, 2017, 29(5): 427-433.
	Cai J, Meng XQ, Dong TT, et al. Progress of plant DEAD-box helicase in response to abiotic stress[J]. Chin Bull Life Sci, 2017, 29(5): 427-433.
[30]	Vandesompele J, de Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biol, 2002, 3(7): 467-470.
[31]	Andersen CL, Jensen JL, Ørntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[J]. Cancer Res, 2004, 64(15): 5245-5250. doi: 10.1158/0008-5472.CAN-04-0496 pmid: 15289330
[32]	Pfaffl MW, Tichopad A, Prgomet C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations[J]. Biotechnol Lett, 2004, 26(6): 509-515. doi: 10.1023/B:BILE.0000019559.84305.47 URL
[33]	吴建阳, 何冰, 杜玉洁, 等. 利用geNorm、NormFinder和BestKeeper软件进行内参基因稳定性分析的方法[J]. 现代农业科技, 2017(5): 278-281.
	Wu JY, He B, Du YJ, et al. Analysis method of systematically evaluating stability of reference genes using geNorm, NormFinder and BestKeeper[J]. Mod Agric Sci Technol, 2017(5): 278-281.
[34]	Xie F, Xiao P, Chen D, et al. miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs[J]. Plant Mol Biol, 2012, 80(1):75-84. doi: 10.1007/s11103-012-9885-2 URL
[35]	Ann M. Regulatory and functional interactions of plant growth regulators and plant glutathione S-transferases(GSTs)[J]. Vitam Horm, 2005, 72: 155-202.
[36]	Agrawal GK, Rakwal R, Jwa NS, et al. Signalling molecules and blast pathogen attack activates rice OsPR1a and OsPR1b genes: a model illustrating components participating during defence/stress response[J]. Plant Physiol Biochem, 2001, 39(12): 1095-1103. doi: 10.1016/S0981-9428(01)01333-X URL
[37]	van Loon LC, Rep M, Pieterse CMJ. Significance of inducible defense-related proteins in infected plants[J]. Annu Rev Phytopathol, 2006, 44: 135-162. pmid: 16602946
[38]	张亚琳, 张亚琨, 江春泉, 等. 人参锈腐病菌Ilyonectria robusta侵染人参根实时定量PCR内参基因的筛选与验证[J/OL]. 吉林农业大学学报, 2022. https://kns.cnki.net/kcms/detail/22.1100.S.20220331.1519.003.html.
	Zhang YL, Zhang YK, Jiang CQ, et al. Screening and verification of internal reference genes in Panax ginseng roots infected by Ilyonectria robusta causing ginseng rusty rot by real-time quantitative PCR[J/OL]. J Jilin Agric Univ, 2022. https://kns.cnki.net/kcms/detail/22.1100.S.20220331.1519.003.html.
[39]	姚李祥, 潘春柳, 余丽莹, 等. 草果种子休眠解除过程中RT-qPCR内参基因筛选[J]. 中国中药杂志, 2021, 46(15): 3832-3837.
	Yao LX, Pan CL, Yu LY, et al. Selection of RT-qPCR reference genes for Amomum tsaoko seeds during dormancy release[J]. China J Chin Mater Med, 2021, 46(15): 3832-3837.
[40]	赵艺蕊, 黄春颖, 王克涛, 等. 山核桃实时荧光定量PCR分析中内参基因的筛选与验证[J]. 果树学报, 2022, 39(1): 10-21.
	Zhao YR, Huang CY, Wang KT, et al. Screening and verification of internal reference genes by real time quantitative PCR analysis in Carya cathayensis[J]. J Fruit Sci, 2022, 39(1): 10-21.
[41]	苏丹丹, 刘玉萍, 张雨, 等. 苦豆子实时荧光定量PCR内参基因筛选与验证[J]. 植物生理学报, 2022, 58(7): 1295-1306.
	Su DD, Liu YP, Zhang Y, et al. Screening and validation of reference genes in Sophora alopecuroides for real-time quantitative PCR[J]. Plant Physiol J, 2022, 58(7): 1295-1306.
[42]	徐圆圆, 赵国春, 郝颖颖, 等. 无患子RT-qPCR内参基因的筛选与验证[J]. 生物技术通报, 2022, 38(10):80-89. doi: 10.13560/j.cnki.biotech.bull.1985.2021-1616
	Xu YY, Zhao GC, Hao YY, et al. Reference genes selection and validation for RT-qPCR in Sapindus mukorossi[J]. Biotechnol Bull, 2022, 38(10):80-89.
[43]	庞强强, 李植良, 罗少波, 等. 高温胁迫下茄子RT-qPCR内参基因筛选及稳定性分析[J]. 园艺学报, 2017, 44(3): 475-486. doi: 10.16420/j.issn.0513-353x.2016-0831
	Pang QQ, Li ZL, Luo SB, et al. Selection and stability analysis of reference gene for RT-qPCR in eggplant under high temperature stress[J]. Acta Hortic Sin, 2017, 44(3): 475-486.
[44]	Faccioli P, Ciceri GP, Provero P, et al. A combined strategy of “in silico” transcriptome analysis and web search engine optimization allows an agile identification of reference genes suitable for normalization in gene expression studies[J]. Plant Mol Biol, 2007, 63(5): 679-688. doi: 10.1007/s11103-006-9116-9 URL
[45]	Liu WG, Tang X, Qi XH, et al. The ubiquitin conjugating enzyme: an important ubiquitin transfer platform in ubiquitin-proteasome system[J]. Int J Mol Sci, 2020, 21(8): 2894. doi: 10.3390/ijms21082894 URL
[46]	Grumati P, Dikic I. Ubiquitin signaling and autophagy[J]. J Biol Chem, 2018, 293(15): 5404-5413. doi: 10.1074/jbc.TM117.000117 pmid: 29187595
[47]	Nakamura N. Ubiquitin system[J]. International Journal of Molecular Sciences, 2018, 19(4):1080. doi: 10.3390/ijms19041080 URL
[48]	Hamey JJ, Wilkins MR. Methylation of elongation factor 1A: where, who, and why?[J]. Trends Biochem Sci, 2018, 43(3): 211-223. doi: S0968-0004(18)30004-5 pmid: 29398204
[49]	Bevitori R, Oliveira MB, Grossi-de-Sá MF, et al. Selection of optimized candidate reference genes for RT-qPCR normalization in rice(Oryza sativa L.)during Magnaporthe oryzae infection and drought[J]. Genet Mol Res, 2014, 13(4): 9795-9805. doi: 10.4238/2014.November.27.7 pmid: 25501189
[50]	代资举, 王艳, 王新涛, 等. 玉米粗缩病诱导下实时荧光定量PCR内参基因的选择[J]. 植物生理学报, 2019, 55(10): 1545-1553.
	Dai ZJ, Wang Y, Wang XT, et al. Reference genes selection for real-time quantitative PCR under rough dwarf virus induced disease in maize[J]. Plant Physiol J, 2019, 55(10): 1545-1553.
[51]	唐枝娟, 刘秦, 肖晓蓉, 等. 白叶枯病菌侵染下的水稻内参基因稳定性[J]. 分子植物育种, 2017, 15(1): 300-306.
	Tang ZJ, Liu Q, Xiao XR, et al. Selection of optimized candidate reference genes for RT-qPCR normalization in rice during Xanthomonas oryzae pv. oryzae infection[J]. Mol Plant Breed, 2017, 15(1): 300-306.
[52]	赵辉, 张春艳, 文艺, 等. 菜豆壳球孢侵染芝麻过程中内参基因的筛选[J]. 中国油料作物学报, 2017, 39(3): 393-398, 419.
	Zhao H, Zhang CY, Wen Y, et al. Screening of reference genes in sesame during Macrophomina phaseolina infection[J]. Chin J Oil Crop Sci, 2017, 39(3): 393-398, 419.
[53]	姚全胜, 杨倩, 柳凤, 等. 芒果细菌性角斑病菌在侵染芒果叶片过程中内参基因筛选[J]. 分子植物育种, 2021, 19(18): 6088-6095.
	Yao QS, Yang Q, Liu F, et al. Screening of reference genes in Xanthomonas citri pv. mangiferaeindicae during the infection of mango leaf[J]. Mol Plant Breed, 2021, 19(18): 6088-6095.
[54]	李恬静薇, 邹潇潇, 朱军, 等. 长茎葡萄蕨藻胁迫条件下RT-qPCR内参基因的筛选与验证[J]. 生物技术通报, 2021, 37(10): 266-276. doi: 10.13560/j.cnki.biotech.bull.1985.2020-1585
	Li T, Zou XX, Zhu J, et al. Selection and validation of reference genes for quantitative real-time PCR in Caulerpa lentillifera under stress conditions[J]. Biotechnol Bull, 2021, 37(10): 266-276.
[55]	陈慧洁, 冯丽贞, 李慧敏, 等. 桉树焦枯病菌内参基因的筛选[J]. 森林与环境学报, 2018, 38(1): 98-103.
	Chen HJ, Feng LZ, Li HM, et al. Screening and evaluation of reference genes of Calonectria pseudoreteaudii[J]. J For Environ, 2018, 38(1): 98-103.

基因名称Gene symbol	基因ID Gene ID	引物序列Primer sequence（5'-3'）	扩增长度Amplified length/bp
UBC1	F02_cb4795_c6/f1p0/677	F：CACTTCCCTCCTGACTATCCCT R：CAAATACTGCCATTGCTGTTGA	92
UBC2	F02_cb10194_c5/f1p0/683	F：AAGGTGCTCCTGTCTATCTGCT R：TGATTCATACTTGGCTCTGTCG	108
UBQ1	F02_cb4331_c39/f4p2/2064	F：GTCAACTCCGTCACCGTAAACA R：GCAACTTCGGACTTCTCCTCTA	119
UBQ2	F02_cb3159_c105/f1p2/1236	F：ACCTTGTGCTCCGTCTTCGTG R：AACATCTGGCTTACCGTCGTCT	162
GAPDH3	F02_cb2521_c25/f1p0/1405	F：GACAAGCCAGTTGCTGTATTCG R：CTTTGCACCACCCTTAATGTGA	140
GAPDH4	F02_cb2521_c44/f7p1/1415	F：AATCACTGCCACCCAGAAGACT R：ACGGAATGACATGCCAGTAAGC	163
TUB	F02_cb8195_c2075/f1p0/1651	F：GGTGGTGGTACTGGATCTGG R：TTGTATGGCTCCACAACTGC	134
EF-1α	F02_cb8884_c10/f1p3/1571	F：ACCGAGTCCATTCCTCCTGTTC R：GGGTTTAGGTGCCTCCTCTTCA	147
40S RP	F02_cb13441_c1/f3p0/558	F：TGACCTCTTGAATCCACCTGC R：AGCAACCTTGGCACTTGACA	107
PABP	F02_cb2343_c66/f1p0/2490	F：TCCTGATATGGCTGGCTTGC R：CCTCGGGCGATTCAAGAAGA	224
DnaJ	F02_cb2629_c9/f1p0/682	F：AGACTCCCTGCAATTCCAGC R：TCTTCATGAACTCCTCGGCG	223
DDX	F02_cb3613_c7/f1p2/2466	F：CCATGTTCAAGAGCAGCACG R：CATGAAGAGCACGAGACCCA	145
eIF-1A	F02_cb13117_c3/f2p1/651	F：GGCTTTGTCATATTCGGGGC R：ATCACCAGCACCATCGTCC	227
GST676	F02_cb3237_c0/f37p2/676	F：GCAAGTCGGTAAAGCTCGGA R：ACTGGCCAAAAGAGTAGCGA	160
PRP902	F02_cb14527_c723/f1p0/902	F：CGTCACAGACAAGCAAAGGC R：CTTACTGTGCCACGTGGGAT	180

基因名称Gene symbol	基因ID Gene ID	引物序列Primer sequence（5'-3'）	扩增长度Amplified length/bp
UBC1	F02_cb4795_c6/f1p0/677	F：CACTTCCCTCCTGACTATCCCT R：CAAATACTGCCATTGCTGTTGA	92
UBC2	F02_cb10194_c5/f1p0/683	F：AAGGTGCTCCTGTCTATCTGCT R：TGATTCATACTTGGCTCTGTCG	108
UBQ1	F02_cb4331_c39/f4p2/2064	F：GTCAACTCCGTCACCGTAAACA R：GCAACTTCGGACTTCTCCTCTA	119
UBQ2	F02_cb3159_c105/f1p2/1236	F：ACCTTGTGCTCCGTCTTCGTG R：AACATCTGGCTTACCGTCGTCT	162
GAPDH3	F02_cb2521_c25/f1p0/1405	F：GACAAGCCAGTTGCTGTATTCG R：CTTTGCACCACCCTTAATGTGA	140
GAPDH4	F02_cb2521_c44/f7p1/1415	F：AATCACTGCCACCCAGAAGACT R：ACGGAATGACATGCCAGTAAGC	163
TUB	F02_cb8195_c2075/f1p0/1651	F：GGTGGTGGTACTGGATCTGG R：TTGTATGGCTCCACAACTGC	134
EF-1α	F02_cb8884_c10/f1p3/1571	F：ACCGAGTCCATTCCTCCTGTTC R：GGGTTTAGGTGCCTCCTCTTCA	147
40S RP	F02_cb13441_c1/f3p0/558	F：TGACCTCTTGAATCCACCTGC R：AGCAACCTTGGCACTTGACA	107
PABP	F02_cb2343_c66/f1p0/2490	F：TCCTGATATGGCTGGCTTGC R：CCTCGGGCGATTCAAGAAGA	224
DnaJ	F02_cb2629_c9/f1p0/682	F：AGACTCCCTGCAATTCCAGC R：TCTTCATGAACTCCTCGGCG	223
DDX	F02_cb3613_c7/f1p2/2466	F：CCATGTTCAAGAGCAGCACG R：CATGAAGAGCACGAGACCCA	145
eIF-1A	F02_cb13117_c3/f2p1/651	F：GGCTTTGTCATATTCGGGGC R：ATCACCAGCACCATCGTCC	227
GST676	F02_cb3237_c0/f37p2/676	F：GCAAGTCGGTAAAGCTCGGA R：ACTGGCCAAAAGAGTAGCGA	160
PRP902	F02_cb14527_c723/f1p0/902	F：CGTCACAGACAAGCAAAGGC R：CTTACTGTGCCACGTGGGAT	180

基因名称 Gene symbol	模拟接菌不同时间的样品 Mock-inoculated samples			接种葱鳞葡萄孢菌不同时间的样品 Samples inoculated with B. squamosa
基因名称 Gene symbol	24 h	48 h	72 h	24 h	48 h	72 h
UBC1	14.63	14.47	15.48	21.21	14.66	21.60
UBC2	45.80	53.35	59.64	72.14	51.73	83.00
UBQ1	8.75	7.90	10.06	12.67	8.00	10.81
UBQ2	187.15	189.18	197.98	223.79	139.12	245.41
GAPDH3	17.86	28.28	23.98	25.81	27.96	15.07
GAPDH4	111.50	168.16	154.12	214.92	132.19	155.86
TUB	49.37	58.77	50.06	58.57	52.65	47.37
EF-1α	28.34	18.20	26.84	48.63	13.83	22.83
40S RP	183.95	252.63	198.60	206.80	235.64	163.86
PABP	87.77	97.44	88.98	97.07	97.91	72.27
DnaJ	516.66	480.78	478.24	429.37	332.07	522.24
DDX	48.00	43.42	35.19	51.43	38.59	33.68
eIF-1A	31.35	44.60	34.58	38.97	33.39	33.45

基因名称 Gene symbol	模拟接菌不同时间的样品 Mock-inoculated samples			接种葱鳞葡萄孢菌不同时间的样品 Samples inoculated with B. squamosa
基因名称 Gene symbol	24 h	48 h	72 h	24 h	48 h	72 h
UBC1	14.63	14.47	15.48	21.21	14.66	21.60
UBC2	45.80	53.35	59.64	72.14	51.73	83.00
UBQ1	8.75	7.90	10.06	12.67	8.00	10.81
UBQ2	187.15	189.18	197.98	223.79	139.12	245.41
GAPDH3	17.86	28.28	23.98	25.81	27.96	15.07
GAPDH4	111.50	168.16	154.12	214.92	132.19	155.86
TUB	49.37	58.77	50.06	58.57	52.65	47.37
EF-1α	28.34	18.20	26.84	48.63	13.83	22.83
40S RP	183.95	252.63	198.60	206.80	235.64	163.86
PABP	87.77	97.44	88.98	97.07	97.91	72.27
DnaJ	516.66	480.78	478.24	429.37	332.07	522.24
DDX	48.00	43.42	35.19	51.43	38.59	33.68
eIF-1A	31.35	44.60	34.58	38.97	33.39	33.45

基因名称 Gene symbol	线性相关系数Linear correlation coefficient（R²）	扩增效率Amplification efficiency/%
UBC1	0.993 9	104.83
UBC2	0.963 0	101.12
UBQ1	0.994 1	110.70
UBQ2	0.990 7	106.64
GAPDH3	0.995 5	109.88
GAPDH4	0.996 6	110.65
EF-1α	0.991 2	110.36
TUB	0.991 7	107.54
40S RP	0.995 3	100.80
DDX	0.990 3	99.19
eIF-1A	0.997 5	106.30
PABP	0.990 9	96.06
DnaJ	0.998 9	106.97