Identification and validation of reference genes for gene expression study using Quantitative Real-Time PCR in shallot (Allium cepa L. Aggregatum group) based on bulb development phase




Abstract. Sulistyaningsih E, Sawitri WD, Handayani VDS, Wicaksono AW, Silalahi CM, Murti RH. 2024. Identification and validation of reference genes for gene expression study using Quantitative Real-Time PCR in shallot (Allium cepa L. Aggregatum group) based on bulb development phase. Biodiversitas 25: 2645-2651. Shallot (Allium cepa L. Aggregatum group) is cultivated vegetatively using bulbs (produce many bulbs) and generatively using TSS (True Seed of Shallot) which tends to produce single bulbs. Bulb aggregation is influenced by activity of genes and the gene expression could be analyzed using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). The qRT-PCR analysis requires reference gene data (housekeeping gene) as a comparison gene against our target gene. This study aimed to select and validate three housekeeping genes in shallot as an initial study for qRT-PCR analysis. The research flow began with the preparation of shallot 'TukTuk' leaves and bulb samples in bulb formation from bulb initial phase to mature phase. Furthermore, a specific primer design based on actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene sequences were obtained from gene bank data. The results showed that the actin, tubulin, and GAPDH genes were successfully amplified and the products were 142 bp, 116 bp, and 136 bp in size, respectively and the genes were expressed almost constantly in leaves and bulbs in all phases of bulb formation. The tubulin gene had the potential as a reference gene for qRT-PCR analysis in shallot with the most consistently expressed.


Antiabong JF, Ngoepe MG, Abechi AS. 2016. Semi-quantitative digital analysis of polymerase chain reaction-electrophoresis gel: Potential applications in low income veterinary laboratories. Veterinary world, 9(9), 935–939. DOI: 10.14202/vetworld.2016.935-939.
Bao W, Qu Y , Shan X, Wan Y. 2016. Screening and Validation of Housekeeping Genes of the Root and Cotyledon of Cunninghamia lanceolata under Abiotic Stresses by Using Quantitative Real-Time PCR. Int. J. Mol. Sci. 17 (1198): 1-17. DOI: 10.3390/ijms17081198.
Blessing CA, Ugrinova GT, Goodson HV. 2004. Actin and ARPs: action in the nucleus. Trends in cell biology. 14(8):435-442. DOI: 10.1016/j.tcb.2004.07.009.
Chen Y, He Q, Li X, Zhang Y, Li J, Zhang L, Yao X , Zhang X , Liu C and Wang H. 2023. Identification of Accurate Reference Genes for qRT-PCR Analysis of Gene Expression in Eremochloa ophiuroides under Multiple Stresses of Phosphorus Deficiency and/or Aluminum Toxicity. Plants 12 (3751):1-13 . DOI: 10.3390/plants12213751.
Dong Z, Zhan B, Li S. 2022. Selection and Validation of Reference Genes for Gene Expression Studies Using Quantitative Real-Time PCR in Prunus Necrotic Ringspot Virus-Infected Cucumis sativus. Viruses. 14(1269): 1-14. DOI: 10.3390/v14061269.
Eisenberg E, Levanon EY. 2013. Human housekeeping genes, revisited. Cell press 29(10): 569-574. DOI: 10.1016/j.tig.2013.05.010.
Gavazzi F, Gaia P, Luca B, Silvia G, Diego B, Laura M. 2017. Evolutionary characterization and transcript profiling of ?-tubulin genes in flax (Linum usitatissimum L.) during plant development. BMC Plant Biol 17 (237): 1-17. DOI 10.1186/s12870-017-1186-0.
Gilliland LU, Pawloski LC, Kandasamy MK, Meagher RB. 2003. Arabidopsis actin gene ACT7 plays an essential role in germination and root growth. The Plant Journal, 33(2), 319-328. DOI: 10.1046/j.1365-313X.2003.01626.x.
Jones RN. 2017. Cytogenetics. In Rabinowitch, H.D. and Brewster J.L. (Eds.), Onions and Allied Crops (Volume I: Botany, Physiology, and Genetics). CRC Press Taylor and Francis Group. DOI: https:10.1201/9781351075169
Kosova AA, Khodyreva SN, Lavrik OI. 2017. Role of glyceraldehyde-3 phosphate dehydrogenase (GAPDH) in DNA repair. Biochemistry. 82(6):643-654. DOI: 10.1134/S0006297917060013.
Lee J , Moon JS, Kim J, Park GO, Kwon JH, Ha IJ, YS, Chang YH. 2020. Evaluation of onion cultivars as affected by bulb maturity and bulb characteristics of intermediateday yellow onions in South Korea. J Hort Sci Biotech 95 (5): 645–660. DOI: 10.1080/14620316.2020.1742586.
Li X, Wei W, Li F, Zhang L, Deng X, Liu Y, Yang S. 2019. The Plastidial Glyceraldehyde-3-Phosphate Dehydrogenase Is Critical for Abiotic Stress Response in Wheat. Int J Mol Sci. 20 (1104): 1-18. DOI: 10.3390/ijms20051104.
Li L, Li N, Fang H, Qi X, Zhou Y. 2020. Selection and Validation of Reference Genes for Normalisation of Gene Expression in Glehnia littoralis. Sci Rep 10 (7374): 1-12. DOI: 10.1038/s41598-020-63917-5
Lim FH, Fakhrana IN, Rasid OA, Idris AS, Parveez GKA, Ho CL, Shaharuddin NA. 2014. Isolation and selection of reference genes for Ganoderma boninense gene expression study using quantitative real-time PCR (qPCR). J Oil Palm Res 26 (2):170–181.
Lin Y, Liu G, Rao Y, Wang B, Tian R , Tan Y , Peng T. 2023. Identification and validation of reference genes for qRT-PCR analyses under different experimental conditions in Allium wallichii. Journal of Plant Physiology 281(153925): 1-9. DOI 10.1016/j.jplph.2023.153925.
Ling H, Wu Q , Guo J, Xu L, Que Y. 2014. Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR. PLoS ONE 9(5): 1-10. DOI:10.1371/journal.pone.0097469.
Maldovan C, Frumuzachi O, Babota? M, Barros L, Mocan A, Carradori S, Crisan G. 2022. Therapeutic Uses and Pharmacological Properties of Shallot (Allium ascalonicum). Front. Nutr 9 : 1-34. DOI: 10.3389/fnut.2022.903686.
Pabuayon IM, Yamamoto N , Trinidad JL, Longkumer T, Raorane ML, Kohli.A. 2016. Reference genes for accurate gene expression analyses across different tissues, developmental stages and genotypes in rice for drought tolerance. Rice 9(32:1-8). DOI 10.1186/s12284-016-0104-7.
Pangestuti R, Sulistyaningsih E, Kurniasih B, Murti RH. 2022. Effect of seed soaking with plant growth regulators combination on the aggregation ability of shallot from seeds. Journal of Agricultural Science 8 (3): 186–195. DOI: 10.22146/ipas.90993.
Radchuk VV. 2008. The Transcriptome of The Tubulin Gene Family In Plants. In: Blume, Y.B., Baird, W.V., Yemets, A.I., Breviario, D. (eds) The Plant Cytoskeleton: a Key Tool for Agro-Biotechnology. NATO Science for Peace and Security Series C: Environmental Security. Springer, Dordrecht. DOI: 10.1007/978-1-4020-8843-8_11.
Rebouças EDL, Costa JJDN, Passos MJ, Passos JRDS, Hurk RVD, Silva, JRV. 2013. Real time PCR and importance of housekeepings genes for normalization and quantification of mRNA expression in different tissues. Brazilian Archives of Biology and Technology, 56(1):143-154. DOI: 10.1590/S1516-89132013000100019.
Rosliani R, Hidayat IM, Sulastrini I, Hilman Y. 2016. Dissemination of technology for shallot (Allium ascalonicum L.) seed production using true shallot seed (TSS) in Indonesia. Acta Hortic 1143:345–351. DOI: 10.17660/ActaHortic.2016.1143.49.
Song J, Cho J, Park J, and Hwang JH. 2022. Identification and validation of stable reference genes for quantitative real time PCR in different minipig tissues at developmental stages. BMC Genomics 23(585): 1-13. DOI: 10.1186/s12864-022-08830-z.
Sulistyaningsih E, Pangestuti R, Rosliani R. 2020. Growth and yield of five prospective shallot selected accessions from true seed of shallot in lowland areas. Journal of Agricultural Science 5 (2): 92-97. DOI: 10.22146/ipas.52457.
Sutardi, Kristamtimi, Purwaningsih H, Widyayanti S, Arianti FD, Pertiwi MD, Triastono J, Praptana RH, Malik A, Cempaka IG, Yusuf, Yufdi MP, Anda M, Wihardjaka A. 2022. Nutrient Management of Shallot Farming in Sandy Loam Soil in Tegalrejo, Gunungkidul, Indonesia. Sustainability 14(19): 1-15. DOI: 10.3390/su141911862.
Tong J, Hu M, Han B, Ji Y, Wang B, Liang H, Liu M, Wu Z, Li N. 2021. Determination of reliable reference for gene expression studies in Chinese chive (Allium tuberosum) based on the transcriptome profiling. Scientific reports 11(16558): 1-12. DOI:10.1038/s41598-021-95849-z
Tsuchiya Y, Nakamura Y, Mikami T. 2021. Japanese Bulb Onion: Production, Consumption, And Cultivars. Journal of Horticultural Research 29(1): 75–82. DOI: 10.2478/johr-2021-0002.
Wang, G., Tian, C., Wang, Y., Wan, F., Hu, L., Xiong, A., & Tian, J. 2019. Selection of reliable reference genes for quantitative RT-PCR in garlic under salt stress. PeerJ, 7: 1-16. DOI: 10.7717/peerj.7319.
Wang, Q., Guo, C., Yang, S., Zhong, Q., & Tian, J. 2023. Screening and Verification of Reference Genes for Analysis of Gene Expression in Garlic (Allium sativum L.) under Cold and Drought Stress. Plants, 12(763): 1-16. DOI: 10.3390/plants12040763.
Valente V, Teixeira SA, Neder L, Okamoto OK, Oba-Shinjo SM, Marie SK, Scrideli CA, Paçó-Larson ML, Carlotti CGJr. 2014. Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR. Ann neurosci 21(2): 62–63. DOI: 10.5214/ans.0972.7531.210207.
Widiastuti A, Sawitri WD, Idris M, Handayani VSD, Winona B, Silalahi CM, Matra DD, Doni F, Setiyadi AH. 2024 (under review). Unraveling the potential UV-B induced gene expression of the primary and secondary metabolisms in shallot. Rev. Agr. Sci. [under review]
Zhang C, Zhang H, Zhan Z, Liu B, Chen Z, Liang Y. 2016. Transcriptome analysis of sucrose metabolism during bulb swelling and development in onion (Allium cepa L.). Front. Plant Sci. 7(1425): 1-11. DOI: 10.3389/fpls.2016.01425.

Most read articles by the same author(s)

1 2 > >>