Analysis of Wax Components and Screening of Wax-deficient Gene Ggl-1 in Garlic （Allium sativum L.）

doi:10.13560/j.cnki.biotech.bull.1985.2025-0220

Abstract

Abstract:

Objective This study is aimed to analyze the wax-deficient components in the leaves of the garlic mutant 8684-gl and identify the wax-deficient gene Ggl-1. The findings may lay foundation for exploring the molecular mechanisms underlying wax formation on garlic leaf surfaces and provide a theoretical basis for garlic pest resistance breeding and its application in agricultural production. Method The garlic wax-deficient mutant 8684-gl and its wild-type counterpart 8684 were used as research materials. Gas Chromatography-Mass Spectrometry (GC-MS) was employed to analyze the wax composition on leaf surfaces. Transcriptome sequencing was conducted to identify the Ggl-1 gene, and its expression was validated using real-time quantitative PCR (RT-qPCR). Result By GC-MS analysis the 39 wax components on the surface of garlic leaves were found, with 16-hentriacontanone (C₃₁H₆₂O) as the primary missing component in 8684-gl. Transcriptome analysis revealed that differentially expressed genes (DEGs) between 8684-gl and wild-type 8684 were mainly involved in lipid transport and metabolism, fatty acid biosynthesis and degradation, and the transport and catabolism of secondary metabolites. Two candidate genes for Ggl-1, Asa8G04000 and Asa4G02100 were identified. RT-qPCR analysis confirmed that these candidate genes presented significant different expressions between 8684-gl and its wild-type counterpart. Conclusion The primary wax-deficient component in 8684-gl was 16-hentriacontanone. The gene Asa8G04000 and Asa4G02100 are identified as candidate genes for the wax-deficient gene Ggl-1, providing insights into the genetic basis of wax biosynthesis in garlic.

Key words: garlic, wax-deficient material, gas chromatograph-mass spectrometer-computer, wax components, transcriptome sequencing, real-time fluorescence quantitative PCR, candidate genes

LIU Ze-zhou, DUAN Nai-bin, YUE Li-xin, WANG Qing-hua, YAO Xing-hao, GAO Li-min, KONG Su-ping. Analysis of Wax Components and Screening of Wax-deficient Gene Ggl-1 in Garlic （Allium sativum L.）[J]. Biotechnology Bulletin, 2025, 41(9): 219-231.

Figures/Tables 11

References 64

[1]	王海平, 宋江萍, 张晓辉, 等. 姜蒜种质资源研究现状及发展建议 [J]. 植物遗传资源学报, 2022, 23(5): 1233-1240.
	Wang HP, Song JP, Zhang XH, et al. Research status and development suggestions for ginger and garlic germplasm resources [J]. J Plant Genet Resour, 2022, 23(5): 1233-1240.
[2]	Lopez-Bellido FJ, Lopez-Bellido RJ, Muñoz-Romero V, et al. New phenological growth stages of garlic (Allium sativum) [J]. Ann Appl Biol, 2016, 169(3): 423-439.
[3]	徐培文, 杨崇良, 崔德才, 等. 大蒜有性生殖技术研究初报 [J]. 园艺学报, 2005, 32(3): 503-506.
	Xu PW, Yang CL, Cui DC, et al. A preliminary study on techniques for sexual reproduction of garlic (Allium sativum L.) [J]. Acta Hortic Sin, 2005, 32(3): 503-506.
[4]	刘冰江, 张海燕, 孔素萍, 等. 大蒜有性生殖研究进展 [J]. 中国蔬菜, 2008(8): 41-44.
	Liu BJ, Zhang HY, Kong SP, et al. Research progress on sexual reproduction of garlic [J]. China Veg, 2008(8): 41-44.
[5]	徐培文, 刘冰江, 孔素萍, 等. 大蒜种质创新和育种研究进展 [J]. 中国蔬菜, 2006(6): 31-33.
	Xu PW, Liu BJ, Kong SP, et al. Research progress on garlic germplasm innovation and breeding [J]. China Veg, 2006(6): 31-33.
[6]	孔素萍, 曹齐卫, 孙敬强, 等. 秋水仙素对大蒜茎尖试管苗四倍体的诱导 [J]. 中国农业科学, 2014, 47(15): 3025-3033.
	Kong SP, Cao QW, Sun JQ, et al. The tetraploid induction of shoot-tip plantlets with colchicine in garlic [J]. Sci Agric Sin, 2014, 47(15): 3025-3033.
[7]	Lewandowska M, Keyl A, Feussner I. Wax biosynthesis in response to danger: its regulation upon abiotic and biotic stress [J]. New Phytol, 2020, 227(3): 698-713.
[8]	Negin B, Shachar L, Meir S, et al. Fatty alcohols, a minor component of the tree tobacco surface wax, are associated with defence against caterpillar herbivory [J]. Plant Cell Environ, 2024, 47(2): 664-681.
[9]	Marcell LM, Beattie GA. Effect of leaf surface waxes on leaf colonization by Pantoea agglomerans and Clavibacter michiganensis [J]. Mol Plant Microbe Interact, 2002, 15(12): 1236-1244.
[10]	Kuźniak E, Gajewska E. Lipids and lipid-mediated signaling in plant-pathogen interactions [J]. Int J Mol Sci, 2024, 25(13): 7255.
[11]	Wang XY, Kong LY, Zhi PF, et al. Update on cuticular wax biosynthesis and its roles in plant disease resistance [J]. Int J Mol Sci, 2020, 21(15): 5514.
[12]	Blenn B, Bandoly M, Küffner A, et al. Insect egg deposition induces indirect defense and epicuticular wax changes in Arabidopsis thaliana [J]. J Chem Ecol, 2012, 38(7): 882-892.
[13]	Camacho-Coronel X, Molina-Torres J, Heil M. Sequestration of exogenous volatiles by plant cuticular waxes as a mechanism of passive associational resistance: a proof of concept [J]. Front Plant Sci, 2020, 11: 121.
[14]	李莉, 赵米贤, 王建华, 等. 植物表皮蜡质合成、运输及调控机制研究进展 [J]. 中国农业大学学报, 2023, 28(7): 1-19.
	Li L, Zhao MX, Wang JH, et al. Research progress on genetic mechanisms of plant epidermal wax synthesis, transport and regulation [J]. J China Agric Univ, 2023, 28(7): 1-19.
[15]	Sajeevan RS. Cuticular waxes and its application in crop improvement[M]//Translating Physiological Tools to Augment Crop Breeding, Springer Nature: Singapore, 2023: 147-176.
[16]	Liu ZZ, Fang ZY, Zhuang M, et al. Fine-mapping and analysis of Cgl1, a gene conferring glossy trait in cabbage (Brassica oleracea L. var. capitata) [J]. Front Plant Sci, 2017, 8: 239.
[17]	Li-Beisson Y, Shorrosh B, Beisson F, et al. Acyl-lipid metabolism [J]. Arabidopsis Book, 2013, 11: e0161.
[18]	Bernard A, Joubès J. Arabidopsis cuticular waxes: Advances in synthesis, export and regulation [J]. Prog Lipid Res, 2013, 52(1): 110-129.
[19]	Sun XD, Zhu SY, Li NY, et al. A chromosome-level genome assembly of garlic (Allium sativum) provides insights into genome evolution and allicin biosynthesis [J]. Mol Plant, 2020, 13(9): 1328-1339.
[20]	Hao F, Liu X, Zhou BT, et al. Chromosome-level genomes of three key Allium crops and their trait evolution [J]. Nat Genet, 2023, 55(11): 1976-1986.
[21]	Chen SF, Zhou YQ, Chen YR, et al. Fastp: an ultra-fast all-in-one FASTQ preprocessor [J]. Bioinformatics, 2018, 34(17): i884-i890.
[22]	Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data [J]. Bioinformatics, 2014, 30(15): 2114-2120.
[23]	Grabherr MG, Haas BJ, Yassour M, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome [J]. Nat Biotechnol, 2011, 29(7): 644-652.
[24]	Davidson NM, Oshlack A. Corset: enabling differential gene expression analysis for de novo assembled transcriptomes [J]. Genome Biol, 2014, 15(7): 410.
[25]	Langmead B, Salzberg SL. Fast gapped-read alignment with bowtie 2 [J]. Nat Methods, 2012, 9(4): 357-359.
[26]	Dobin A, Davis CA, Schlesinger F, et al. STAR: ultrafast universal RNA-seq aligner [J]. Bioinformatics, 2013, 29(1): 15-21.
[27]	Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome [J]. BMC Bioinformatics, 2011, 12: 323.
[28]	Tarazona S, Furió-Tarí P, Turrà D, et al. Data quality aware analysis of differential expression in RNA-seq with NOISeq R/Bioc package [J]. Nucleic Acids Res, 2015, 43(21): e140.
[29]	Chen TT, Chen X, Zhang SS, et al. The genome sequence archive family: toward explosive data growth and diverse data types [J]. Genom Proteom Bioinform, 2021, 19(4): 578-583.
[30]	Kõressaar T, Lepamets M, Kaplinski L, et al. Primer3_masker: integrating masking of template sequence with primer design software [J]. Bioinformatics, 2018, 34(11): 1937-1938.
[31]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-△△CT method [J]. Methods, 2001, 25(4): 402-408.
[32]	González-Valenzuela L, Renard J, Depège-Fargeix N, et al. The plant cuticle [J]. Curr Biol, 2023, 33(6): R210-R214.
[33]	Hannoufa A, McNevin J, Lemieux B. Epicuticular waxes of eceriferum mutants of Arabidopsis thaliana [J]. Phytochemistry, 1993, 33(4): 851-855.
[34]	Bourdenx B, Bernard A, Domergue F, et al. Overexpression of Arabidopsis ECERIFERUM1 promotes wax very-long-chain alkane biosynthesis and influences plant response to biotic and abiotic stresses [J]. Plant Physiol, 2011, 156(1): 29-45.
[35]	Gozdzik J, Busta L, Jetter R. Leaf cuticular waxes of wild-type Welsh onion (Allium fistulosum L.) and a wax-deficient mutant: Compounds with terminal and mid-chain functionalities [J]. Plant Physiol Biochem, 2023, 198: 107679.
[36]	Zhu MZ, Xing JY, Wang YQ. Transcriptome and metabolome integrated analysis reveals the wax biosynthesis mechanism of Allium cepa L [J]. Sci Hortic, 2024, 333: 113251.
[37]	Munaiz ED, Townsend PA, Havey MJ. Reflectance spectroscopy for non-destructive measurement and genetic analysis of amounts and types of epicuticular waxes on onion leaves [J]. Molecules, 2020, 25(15): 3454.
[38]	Liu XN, Gao S, Liu Y, et al. Comparative analysis of the chemical composition and water permeability of the cuticular wax barrier in Welsh onion (Allium fistulosum L.) [J]. Protoplasma, 2020, 257(3): 833-840.
[39]	Chemelewski R, McKinley BA, Finlayson S, et al. Epicuticular wax accumulation and regulation of wax pathway gene expression during bioenergy sorghum stem development [J]. Front Plant Sci, 2023, 14: 1227859.
[40]	Wójcicka A. Effect of epicuticular waxes from Triticale on the feeding behaviour and mortality of the grain aphid, Sitobion avenae (Fabricius) (Hemiptera: Aphididae) [J]. J Plant Prot Res, 2016, 56(1): 39-44.
[41]	Gorb E, Haas K, Henrich A, et al. Composite structure of the crystalline epicuticular wax layer of the slippery zone in the pitchers of the carnivorous plant Nepenthes alata and its effect on insect attachment [J]. J Exp Biol, 2005, 208(Pt 24): 4651-4662.
[42]	Cardona JB, Grover S, Bowman MJ, et al. Sugars and cuticular waxes impact sugarcane aphid (Melanaphis sacchari) colonization on different developmental stages of sorghum [J]. Plant Sci, 2023, 330: 111646.
[43]	Gorb EV, Gorb SN. Anti-adhesive effects of plant wax coverage on insect attachment [J]. J Exp Bot, 2017, 68(19): 5323-5337.
[44]	Cardona JB, Grover S, Busta L, et al. Sorghum cuticular waxes influence host plant selection by aphids [J]. Planta, 2022, 257(1): 22.
[45]	Lei PZ, Pan MH, Kang SQ, et al. A premature termination codon mutation in the onion AcCER2 gene is associated with both glossy leaves and thrip resistance [J]. Hortic Res, 2025, 12(4): uhaf006.
[46]	Xu XD, Chen Y, Li BQ, et al. Molecular mechanisms underlying multi-level defense responses of horticultural crops to fungal pathogens [J]. Hortic Res, 2022, 9: uhac066.
[47]	Ma PP, Liu EP, Zhang ZR, et al. Genetic variation in ZmWAX2 confers maize resistance to Fusarium verticillioides [J]. Plant Biotechnol J, 2023, 21(9): 1812-1826.
[48]	Liu RL, Zhang LP, Xiao SY, et al. Ursolic acid, the main component of blueberry cuticular wax, inhibits Botrytis cinerea growth by damaging cell membrane integrity [J]. Food Chem, 2023, 415: 135753.
[49]	Jiang YY, Peng YT, Hou GY, et al. A high epicuticular wax strawberry mutant reveals enhanced resistance to Tetranychus urticae Koch and Botrytis cinerea [J]. Sci Hortic, 2024, 324: 112636.
[50]	Ziv C, Zhao ZZ, Gao YG, et al. Multifunctional roles of plant cuticle during plant-pathogen interactions [J]. Front Plant Sci, 2018, 9: 1088.
[51]	Yu YB, Zhang S, Yu Y, et al. The pivotal role of MYB transcription factors in plant disease resistance [J]. Planta, 2023, 258(1): 16.
[52]	Lee SB, Suh MC. Regulatory mechanisms underlying cuticular wax biosynthesis [J]. J Exp Bot, 2022, 73(9): 2799-2816.
[53]	Xu LP, Hao JX, Lv MF, et al. A genome-wide association study identifies genes associated with cuticular wax metabolism in maize [J]. Plant Physiol, 2024, 194(4): 2616-2630.
[54]	Jian L, Kang K, Choi Y, et al. Mutation of OsMYB60 reduces rice resilience to drought stress by attenuating cuticular wax biosynthesis [J]. Plant J, 2022, 112(2): 339-351.
[55]	He JJ, Li CZ, Hu N, et al. ECERIFERUM1-6A is required for the synthesis of cuticular wax alkanes and promotes drought tolerance in wheat [J]. Plant Physiol, 2022, 190(3): 1640-1657.
[56]	Zhu L, Guo JS, Zhu J, et al. Enhanced expression of EsWAX1 improves drought tolerance with increased accumulation of cuticular wax and ascorbic acid in transgenic Arabidopsis [J]. Plant Physiol Biochem, 2014, 75: 24-35.
[57]	Wang Y, Wang ML, Sun YL, et al. Molecular characterization of TaFAR1 involved in primary alcohol biosynthesis of cuticular wax in hexaploid wheat [J]. Plant Cell Physiol, 2015, 56(10): 1944-1961.
[58]	Wang Y, Wang ML, Sun YL, et al. FAR5, a fatty acyl-coenzyme A reductase, is involved in primary alcohol biosynthesis of the leaf blade cuticular wax in wheat (Triticum aestivum L.) [J]. J Exp Bot, 2015, 66(5): 1165-1178.
[59]	Kan Y, Mu XR, Zhang H, et al. TT2 controls rice thermotolerance through SCT1-dependent alteration of wax biosynthesis [J]. Nat Plants, 2022, 8(1): 53-67.
[60]	Wang ZY, Tian XJ, Zhao QZ, et al. The E3 ligase DROUGHT HYPERSENSITIVE negatively regulates cuticular wax biosynthesis by promoting the degradation of transcription factor ROC4 in rice [J]. Plant Cell, 2018, 30(1): 228-244.
[61]	Xiong C, Xie QM, Yang QH, et al. WOOLLY, interacting with MYB transcription factor MYB31, regulates cuticular wax biosynthesis by modulating CER6 expression in tomato [J]. Plant J, 2020, 103(1): 323-337.
[62]	Tang J, Yang XP, Xiao CL, et al. GDSL lipase occluded stomatal pore 1 is required for wax biosynthesis and stomatal cuticular ledge formation [J]. New Phytol, 2020, 228(6): 1880-1896.
[63]	Zhao HY, Zhang HM, Cui P, et al. The putative E3 ubiquitin ligase ECERIFERUM9 regulates abscisic acid biosynthesis and response during seed germination and postgermination growth in Arabidopsis [J]. Plant Physiol, 2014, 165(3): 1255-1268.
[64]	Wu P, Gao HN, Liu J, et al. Insight into the roles of the ER-associated degradation E3 ubiquitin ligase HRD1 in plant cuticular lipid biosynthesis [J]. Plant Physiol Biochem, 2021, 167: 358-365.

样品名Sample	原始读段数目 Raw reads （Mb）	过滤读段数目 Clean reads （Mb）	过滤后数据量 Clean data （Gb）	Q30质量值比例Q30 rate （%）	比对率Alignment rate （%）	测序年份Sequencing batch
GL1A	45.44	42.38	6.36	92.49	93.27	2023
GL2A	45.44	42.54	6.38	92.94	93.62
GL3A	45.44	42.03	6.30	93.50	92.50
GY1A	45.44	42.74	6.41	93.17	94.06
GY2A	45.44	42.41	6.36	93.81	93.35
GY3A	45.44	42.49	6.37	93.19	93.52
Ggl-1	62.61	61.63	9.25	94.53	88.86	2022
Ggl-2	74.48	71.80	10.77	92.58	88.86
Ggl-3	73.45	70.40	10.56	92.52	88.39
Gwx-1	68.59	65.25	9.79	92.91	89.24
Gwx-2	56.61	55.72	8.36	93.85	88.93
Gwx-3	71.67	68.79	10.32	92.90	87.89

样品名Sample	原始读段数目 Raw reads （Mb）	过滤读段数目 Clean reads （Mb）	过滤后数据量 Clean data （Gb）	Q30质量值比例Q30 rate （%）	比对率Alignment rate （%）	测序年份Sequencing batch
GL1A	45.44	42.38	6.36	92.49	93.27	2023
GL2A	45.44	42.54	6.38	92.94	93.62
GL3A	45.44	42.03	6.30	93.50	92.50
GY1A	45.44	42.74	6.41	93.17	94.06
GY2A	45.44	42.41	6.36	93.81	93.35
GY3A	45.44	42.49	6.37	93.19	93.52
Ggl-1	62.61	61.63	9.25	94.53	88.86	2022
Ggl-2	74.48	71.80	10.77	92.58	88.86
Ggl-3	73.45	70.40	10.56	92.52	88.39
Gwx-1	68.59	65.25	9.79	92.91	89.24
Gwx-2	56.61	55.72	8.36	93.85	88.93
Gwx-3	71.67	68.79	10.32	92.90	87.89

基因 Gene	正向引物序列 Forward sequence （5′-3′）	反向引物序列 Reverse sequence （5′-3′）
AsACTIN	TCCTAACCGAGCGAGGCTACAT	GGAAAAGCACTTCTGGGCACC
Asa8G04000	CACTGCTGCTGTGCTTTCAG	ACTGGGACGATCTTCCTGGA
Asa4G02100	TGAGGTGATGCCAGTGAACC	TCACCCAGTCGAAATGTGCA
Asa7G01089	CGCTGGTAGTCCACACCTTT	TAACGTGTCCAAGCTCCCAC
Asa5G03876	TTGGCAAGAGTAGCAGCTCC	GATCGAATAGCGACTGGCCA
Asa7G02324	GTCCTCTGGAATTGCTGCCT	CGCCTTCATACGCCCATTTG
Asa4G04412	AGCGCTGGAGTTGGAGTTAC	ATGTTCATCACAGCCCCTGG
Asa1G04660	TGTAGGATCTCGTTCGGGGT	GACGACAGGGTTTACAGCCA
Asa1G03601	CCTGAGTTCGTCGATACCGG	TCCATAAACATGCACCCGCT
Asa1G03571	TGAAGCGTCGCAAAACTGTG	TTTCTCCGGGGCACTTCATC
Asa4G06138	GGAAGCTTCCCATGTTCGGA	ACGGACGTTGTCAAATCCGA
Asa2G01695	TTAACCGCTTGTTTGCACCG	TAGTCAACAGCCAGCATGCA
Asa0G03810	AGGGCATCCTTGGGGAAATG	TGTTCGGGACTCCATTTGCA

基因 Gene	正向引物序列 Forward sequence （5′-3′）	反向引物序列 Reverse sequence （5′-3′）
AsACTIN	TCCTAACCGAGCGAGGCTACAT	GGAAAAGCACTTCTGGGCACC
Asa8G04000	CACTGCTGCTGTGCTTTCAG	ACTGGGACGATCTTCCTGGA
Asa4G02100	TGAGGTGATGCCAGTGAACC	TCACCCAGTCGAAATGTGCA
Asa7G01089	CGCTGGTAGTCCACACCTTT	TAACGTGTCCAAGCTCCCAC
Asa5G03876	TTGGCAAGAGTAGCAGCTCC	GATCGAATAGCGACTGGCCA
Asa7G02324	GTCCTCTGGAATTGCTGCCT	CGCCTTCATACGCCCATTTG
Asa4G04412	AGCGCTGGAGTTGGAGTTAC	ATGTTCATCACAGCCCCTGG
Asa1G04660	TGTAGGATCTCGTTCGGGGT	GACGACAGGGTTTACAGCCA
Asa1G03601	CCTGAGTTCGTCGATACCGG	TCCATAAACATGCACCCGCT
Asa1G03571	TGAAGCGTCGCAAAACTGTG	TTTCTCCGGGGCACTTCATC
Asa4G06138	GGAAGCTTCCCATGTTCGGA	ACGGACGTTGTCAAATCCGA
Asa2G01695	TTAACCGCTTGTTTGCACCG	TAGTCAACAGCCAGCATGCA
Asa0G03810	AGGGCATCCTTGGGGAAATG	TGTTCGGGACTCCATTTGCA

材料Material	株高Plant height （cm）	假茎高Pseudostem height （cm）	叶长Leaf length （cm）	叶宽Leaf width （cm）	茎粗Stem thick （cm）
8684	92.40±3.78	36.95±1.86	58.70±2.70	3.64±0.23	18.08±1.59
8684-gl	94.55±2.83	37.75±1.86	59.60±2.50	3.46±0.20	17.21±1.01