Novel methylation specific bisulfite primer pairs for epigenetic studies of Capsicum spp.

Year 2023, Volume: 7 Issue: 4, 918 - 925, 29.12.2023

Ayse Gul Ince , Mehmet Karaca

https://doi.org/10.31015/jaefs.2024.4.23

Abstract

Over the past ten years, interest in epigenetic has rapidly increased. Heritable and stable changes in gene expression without any change in DNA sequence is in the field of epigenetics. Plants have a well-preserved epigenetic signature called DNA methylation. It is an essential epigenetic mark that protects genomic stability, silences harmful transposon insertions, and controls global gene expression in all developmental stages and environmental circumstances. All three sequence contexts, the asymmetric CpHpH context and the symmetric CpG and CpHpG contexts (where H is C, A, or T), are among DNA methylation sites in plants. Particularly, DNA cytosine methylation affects a wide range of biological processes, such as gene expression, chromatin structure, DNA packing, recombination, genomic imprinting, and DNA replication. The choice of primer pairs that flank cytosine methylation contexts is critical when designing for the detection of DNA cytosine methylation using bisulfite sequencing. We have developed and synthesized 26 bisulfite specific primer pairs suitable for DNA cytosine methylation investigations in peppers. These primers are specific to certain promoters, intergenic regions, and gene bodies (exons, introns, and UTRs). DNA samples taken from various tissues and developmental stages of Capsicum annuum L. Demre Sivrisi were analyzed by these primer pairs to confirm their utilization.

Keywords

Cytosine methylation, Epigenetics, Exon, Intron, Promoter

Ethical Statement

Ethics committee approval is not required.

Supporting Institution

TÜBİTAK

Project Number

113O935

Thanks

This study was financially supported by a grant from The Scientific and Technological Research Council of Turkey (Project No: 113O935).

References

• Araz, O., Ekinci, M., Yüce, M., Shams, M., Ağar, G., & Yildirim, E., (2022). Low-temperature modified DNA methylation level, genome template stability, enzyme activity, and proline content in pepper (Capsicum annuum L.) genotypes. Scientia Horticulturae, 294, 110761. https://doi.org/10.1016/j.scienta.2021.110761
• Cai, Y., Xu, M., Liu, J., Zeng, H., Song, J., Sun, B., Chen, S., Deng, Q., Lei, J., Cao, B., Chen, C., Chen, M., Chen, K., Chen, G., & Zhu, Z. (2022) Genome-wide analysis of histone acetyltransferase and histone deacetylase families and their expression in fruit development and ripening stage of pepper (Capsicum annuum). Frontiers in Plant Science, 13, 971230. https://doi.org/10.3389/fpls.2022.971230
• Dhingra, T., Mittal, K., & Sarma, G. S. (2014). Analytical techniques for DNA methylation - An Overview. Current Pharmaceutical Analysis, 10: 71-85.
• Gallusci, P., Hodgman, C., Teyssier, E., & Seymour, G. B. (2016). DNA methylation and chromatin regulation during fleshy fruit development and ripening. Frontiers Plant Science, 7, 807.
• Gruntman, E., Qi, Y., Slotkin, R. K., Roeder, T., Martienssen R. A., & Sachidanandam, R. (2008). Kismeth: analyzer of plant methylation states through bisulfite sequencing. BMC Bioinformatics, 9: 371.
• Hulse-Kemp, A. M., Maheshwari, S., Stoffel, K., Hill, T. A., Jaffe, D., Williams, S. R., et al. (2018). Reference quality assembly of the 3.5-Gb genome of Capsicum annuum from a single linked-read library. Horticulture Research, 5(4), 10. https://doi.org/1038/s41438-017-0011-0
• Ince, A. G., Karaca, M., & Onus, A. N. (2010a). Genetic relationships within and between Capsicum species. Biochemical Genetics. 48, 83-95.
• Ince, A. G., Karaca, M., & Onus, A. N. (2010b). Differential expression patterns of genes containing microsatellites in Capsicum annuum L. Molecular Breeding, 25, 645-658.
• Ince, A. G., & Karaca, M. (2017). Development and utilization of bisulfite specific primer pairs for epigenetic studies in Solanum lycopersicum L. Journal of Scientific and Engineering Research, 4(11), 145-153.
• Ince, A. G., & Karaca, M. (2021). Tissue and/or developmental stage specific methylation of nrDNA in Capsicum annuum. Journal of Plant Research, 134(4), 841-855. https://doi.org/10.1007/s10265-021-01287-3
• Ince, A. G., & Karaca, M. (2009). The MAGi RNA extraction method: highly efficient and simple procedure for fresh and dry plant tissues. Journal of the Science of Food and Agriculture, 89, 168-176.
• Ince, A. G., Yildiz, F., & Karaca, M. (2011). The MAGi DNA extraction method for fresh tissues and dry seeds. Journal of Medicinal Plants Research, 5, 5458-5464.
• Ince, A. G., Karaca, M., & Onus, A. N. (2011). Exact microsatellite density differences among Capsicum tissues and development stages. Journal of Agricultural Sciences, 17, 291‐299.
• Jaiswal, V., Rawoof, A., Gahlaut, V., Ahmad, I., Chhapekar, S. S., Dubey, M., & Ramchiary, N. (2022). Integrated analysis of DNA methylation, transcriptome, and global metabolites in interspecific heterotic Capsicum F1 hybrid. iScience, 25(11), 105318. https://doi.org/10.1016/j.isci.2022.105318
• Jin, X., Pang, Y., Jia, F., Xiao, G., Li, Q., & Zhu, Y. (2013). A potential role for CHH DNA methylation in cotton fiber growth patterns. PLoS ONE, 8, e60547.
• Karaca, M., Ince, A. G., Elmasulu, S. Y., Onus, A. N., & Turgut, K. (2005). Coisolation of genomic and organelle DNAs from 15 genera and 31 species of plants. Analytical Biochemistry, 343, 353-355.
• Karaca, M., & Ince A. G. (2011). New non-redundant microsatellite and CAPS-microsatellite markers for cotton (Gossypium L.). Turkish Journal of Field Crops, 16, 172-178.
• Karaca, M., & Ince, A. G. (2016). EpiOne: A software tool for identification of potential cytosine DNA methylation marks in promoters and gene bodies. Journal of Scientific and Engineering Research, 3, 295-301.
• Karaca, M., Ince, A. G., Uygur-Gocer, E., & Aydin, A. (2016a). Bisulfite primer pairs for analysis of cotton (Gossypium spp.) DNA methylation. Journal of Scientific and Engineering Research, 3, 656-662.
• Karaca, M., Ince, A. G., Uygur-Gocer, E., & Aydin, A. (2016b). Exonic and intronic DNA methylation differences in a fiber specific gene of Pima cotton (Gossypium barbadense L.). Journal of Scientific and Engineering Research, 3, 478-486.
• Kim, G., Clarke, C. R., Larose, H., Tran, H. T., Haak, D. C., Zhang L., et al., (2017). Herbicide injury induces DNA methylome alterations in Arabidopsis. PeerJ, 5, e3560. https://doi.org/10.7717/peerj.3560
• Kim, S., Park, M., Yeom, SI. et al. (2014). Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nature Genetics, 46, 270-278 https://doi.org/10.1038/ng.2877
• Liu, F., Yu, H., Deng, Y., Zheng, J., Liu, M., Ou, L., et al., (2017). PepperHub, an informatics hub for the Chili pepper research community. Molecular Plant, 10, 1129-1132. https://doi.org/10.1016/j.molp.2017.03.005
• Mazourek, M., Pujar, A., Borovsky, Y., Paran, I., Mueller, L., & Jahn, M. M. (2009) A dynamic interface for capsaicinoid systems biology. Plant Physiology, 150, 1806-1821.
• Paran, I., & van der Knaap, E. (2007). Genetic and molecular regulation of fruit and plant domestication traits in tomato and pepper. Journal of Experimental Botany, 58, 3841-3852. https://doi.org/10.1093/jxb/erm257
• Peng, H., & Zhang, J. (2009). Plant genomic DNA methylation in response to stresses: potential applications and challenges in plant breeding. Progress in Natural Science, 19, 1037-1045.
• Portis, E., Acquadro, A., Comino, C., & Lanteri, S. (2004). Analysis of DNA methylation during germination of pepper (Capsicum annuum L.) seeds using methylation sensitive amplification polymorphism (MSAP). Plant Science, 166, 1.
• Qin, C., Yu, C., Shen, Y., Fang, X., Chen, L., Min, J., Cheng, J., Zhao, S., Xu, M., Luo, Y., et al. (2014). Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization. Proceedings of the National Academy of Sciences, 111, 5135-5140. https://doi.org/10.4172/2168-9881.s1.013
• Rand, K. N., Mitchell, S. M., Clark, S. J., & Molloy, P. L. (2006). Bisulphite differential denaturation PCR for analysis of DNA methylation. Epigenetics, 1, 94-100.
• Shams, M., Yildirim, E., Arslan, E. et al. (2020). Salinity induced alteration in DNA methylation pattern, enzyme activity, nutrient uptake and H2O2 content in pepper (Capsicum annuum L.) cultivars. Acta Physiologiae Plantarum, 42, 59. https://doi.org/10.1007/s11738-020-03053-9
• Smulders, M. J. M., & de Klerk, G. J. (2011). Epigenetics in plant tissue culture. Plant Growth Regulation, 63, 137-146.
• Teyssier, E., Bernacchia, G., Maury, S., HowKit, A., Stammitti-Bert, L., Rolin, D., et al., (2008). Tissue dependent variations of DNA methylation and endoreduplication levels during tomato fruit development and ripening. Planta, 228, 391-399.
• Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M., & Rozen, S. G. (2012). Primer3--new capabilities and interfaces. Nucleic Acids Research, 1, 40(15), e115.
• Warnecke, P. M., Stirzaker, C., Melki, J. R., Millar, D. S., Paul, C. L., & Clark, S. J. (1997). Detection & measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA. Nucleic Acids Research, 25, 4422-4426.
• Xiao, K., Chen, J., He, Q., Wang, Y., Shen, H., & Sun, L. (2020). DNA methylation is involved in the regulation of pepper fruit ripening and interacts with phytohormones. Journal of Experimental Botany, 25, 71(6), 1928-1942. https://doi.org/10.1093/jxb/eraa003
• Xu, X. W., Li, T., Li, Y., Wang, H. M. (2015). Variation of DNA cytosine methylation patterns among parent lines and reciprocal hybrids in hot pepper. Chemical Engineering Transactions, 46, 1345-1350.