Improved Performance of SiDREB2-SNAP Marker in Foxtail Millet by Optimum Primer Concentration, PCR Cycle, and DNA Polymerase Specificity
DOI:
https://doi.org/10.29244/jtcs.13.01.103-113Keywords:
abiotic stress, allele-specific marker, cereals, drought, molecular marker, protocolAbstract
Foxtail millet (Setaria italica L. Beauv.) is an emerging carbohydrate-producing crop. It is considered a climate-resilient crop due to its tolerance to abiotic stresses. Breeding for abiotic-tolerant crops requires powerful tools such as molecular markers. The single-nucleotide amplified polymorphism (SNAP) marker, developed from a single-nucleotide DNA variation at a specific location in the plant genome, is a simple yet powerful PCR-based marker widely used in phenotype-related selection. A SiDREB2-based SNAP marker was previously developed based on a base variation at the 558th base pair (an A/G transition) in the SiDREB2 gene and used to estimate the drought tolerance in foxtail millet. However, the specificity of the marker depends on technical aspects, including the type of DNA polymerase used, primer concentration, and the number of PCR cycles. Here, we reported that non-specific and false positive amplicons can be eliminated by utilizing DNA polymerase with no 3’ to 5’ proofreading activity and reducing the final primer concentration to 1.25 μM. PCR cycle number 25 yielded the optimum specificity, while increasing the cycle to 30 resulted in false positive results. Altogether, our results showed that technical optimization is necessary for improving the specificity of the SNAP marker.
References
Aboul-Maaty, N. A. F., & Oraby, H. A. S. (2019). Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Bulletin of the National Research Centre, 43, 25. https://doi.org/10.1186/s42269-019-0066-1
Ardie, S. W., Nugroho, R. B., Dirpan, A., & Anshori, M. F. (2025). Foxtail millet research in supporting climate change resilience efforts: Bibliometric analysis and focused literature review. Heliyon, 11(3), e42348. https://doi.org/10.1016/j.heliyon.2025.e42348
Arora, L., Aggarwal, R., Dhaliwal, I., Gupta, O. P., & Kaushik, P. (2023). Assessment of sensory and nutritional attributes of foxtail millet-based food products. Frontiers Nutrition, 10, 1146545. https://doi.org/10.3389/fnut.2023.1146545
Asif, S., Khan, M., Arshad, M. W., & Shabbir, M. I. (2021). PCR optimization for beginners: A step by step guide. Research in Molecular Medicine, 9(2), 81–102. http://dx.doi.org/10.32598/rmm.9.2.1189.1
Bhardwaj, T., Rathore, A. S., & Jha, S. K. (2020). The selection of highly specific and selective aptamers using modified SELEX and their use in process analytical techniques for Lucentis bioproduction. RSC Advances, 10(48), 28906–28917. https://doi.org/10.1039/D0RA03542D
Butarbutar, L. K. S., Syawaluddin, D. D., Suwarno, W. B., & Ardie, S. W. (2024). SiDREB2-based SNAP marker-assisted and multi-trait selection in the early generation of foxtail millet (Setaria italica L. Beauv.). Journal of Tropical Biodiversity and Biotechnology, 9(3), jtbb90407. https://doi.org/10.22146/jtbb.90407
Drenkard, E., Richter, B. G., Rozen, S., Stutius, L. M., Angell, N. A., Mindrinos, M., Cho, R.J ., Oefner, P. J., Davis, R. W., & Ausubel, F. M. (2000). A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis. Plant Physiology, 124(4),1483-1492. https://doi.org/10.1104/pp.124.4.1483
Gaudet M., Fara A. G., Beritognolo I., & Sabatti M. (2009). Allele-Specific PCR in SNP Genotyping. In A. Komar (Ed.), Single Nucleotide Polymorphisms vol 578 Methods in Molecular Biology™ (pp 415–424). Humana Press. https://doi.org/10.1007/978-1-60327-411-1_26
Ghashghaei, S., Etemadifar, Z., Tavassoli, M., & Mofid, M. R. (2023). Optimization of degenerate PCR conditions for reducing error rates in detection of PKS and NRPS gene groups in actinomycetes. Avicenna Journal of Medical Biotechnology, 15(1), 28. https://doi.org/10.18502/ajmb.v15i1.11422
Grigorieva, E., Livenets, A., & Stelmakh, E. (2023). Adaptation of agriculture to climate change: A scoping review. Climate, 11(10), 202. https://doi.org/10.3390/cli11100202
Hasan, N., Choudhary, S., Naaz, N., Sharma, N., & Laskar, R. A. (2021). Recent advancements in molecular marker assisted selection and applications in plant breeding programmes. Journal of Genetic Engineering and Biotechnology, 19(1), 128. https://doi.org/10.1186/s43141-021-00231-1
Hasegawa, T., Wakatsuki, H., Ju, H., Vyas, S., Nelson, G. C., Farrell, A., Deryng, D., Meza, F., & Makowski, D. (2022). A global dataset for the projected impacts of climate change on four major crops. Scientific Data. 9(1), 58. https://doi.org/10.1038/s41597-022-01150-7
Hatta, A. N. N. L., Sukma, D., Maskromo, I., & Sudarsono, S. (2023). Validated SNAP markers based on the CYP P450 87 A3 gene in coconut (Cocos nucifera) are associated with yearly stem height increase. Biodiversitas, 24(5), 2503–2512. https://doi.org/10.13057/biodiv/d240501
Jang, Y. E. & Lee, S. (2021). Gene-based allele specific marker for resistance to Phytophthora sojae in soybean (Glycine max L.). Plant Breeding and Biotechnology, 9(2), 164–169. https://doi.org/10.9787/PBB.2021.9.2.164
Kim, M. Y., Van, K., Lestari, P., Moon, J. K., & Lee, S. H. (2005). SNP identification and SNAP marker development for a GmNARK gene controlling supernodulation in soybean. Theoretical and Applied Genetics, 110,1003–1010. https://doi.org/10.1007/s00122-004-1887-2
Lata, C., Bhutty, S., Bahadur, R. P., Majee, M., & Prasad, M. (2011). Association of an SNP in a novel DREB2-like gene SiDREB2 with stress tolerance in foxtail millet [Setaria italica (L.)]. Journal of Experimental Botany, 62(10), 3387–3401. https://doi.org/10.1093/jxb/err016
Laura, S. L., & Rukmanidevi, D. (2023). A review of PCR, principle, and its applications. International Journal of Pharmaceutical Research and Applications, 8(3), 3502 3513.
Liaqat, W., Barutçular, C., Farooq, M. U., Ahmad, H., Jan, M. F., Ahmad, Z., Nawaz, H., & Li, M. (2022). Climate change in relation to agriculture: A review. Spanish Journal of Agricultural Research, 20(2), e03R01. https://doi.org/10.5424/sjar/2022202-17742
Liu, J., Huang, S., Sun, M., Liu, S., Liu, Y., Wang, W., Zhang, X., Wang, H., & Hua, W. (2012). An improved allele-specific PCR primer design method for SNP marker analysis and its application. Plant Methods, 8(34). https://doi.org/10.1186/1746-4811-8-34
Lorenz, T. C. (2012). Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. JoVE Journal, 63, e3998. https://doi.org/10.3791/3998
Mubarak, S. M., Al-Koofee, D. A., Radhi, O. A., Ismael, J. M., & Al-Zubaidi, Z. F. (2020). An optimization and common troubleshooting solving in polymerase chain reaction technique. Systematic Reviews in Pharmacy, 11(2), 427-436. https://doi.org/10.5530/srp.2020.2.63
Mubarok, A. F., Purwantomo, S., Suwanto, A., Reflinur., & Ardie, S. W. (2025). SNAP markers for pyramided yield and BLB resistance genes in rice. Crop Breeding and Applied Biotechnology, 25(4), e534425411.
Muzzayyanah, P. N., Suwarno, W. B., & Ardie, S. W. (2024). Gene action and heritability estimates in F2 populations of foxtail millet (Setaria italica L.). SABRAO Journal of Breeding and Genetics, 56(1), 65–75. http://doi.org/10.54910/sabrao2024.56.1.6
Nazir, I., & Mahmood, H. Z. (2020). Polymerase chain reaction: A creative review. Journal of Applied Biotechnology and Bioengineering, 7(4),157–159. https://doi.org/10.15406/jabb.2020.07.00228
Nugroho, R. B., Suwarno, W. B., Khumaida, N., & Ardie, S. W. (2020). Male-sterile induction method in foxtail millet (Setaria italica). Biodiversitas, 21(9), 4325–4330. https://doi.org/10.13057/biodiv/d210951
Nugroho, R. B., Chusnah, U., Suwarno, W. B., & Ardie, S. W. (2024). Flowering phenology and SiDREB2-based SNAP marker-assisted hybridity confirmation for artificial hybridization of Indonesian foxtail millet (Setaria italica L. Beauv) genotypes. HAYATI Journal of Biosciences, 31(6), 1082–1094. https://doi.org/10.4308/hjb.31.6.1082-1094
Ratnawati, S., Jannah, R. M., Dewi, Y. I., Rizqullah, R., Suwarno, W. B., & Ardie, S. W. (2024). The genetic variability of Indonesian local foxtail millet accession based on agro-morphological traits and early salinity tolerance evaluation utilizing SiDREB2-based SNAP marker. HAYATI Journal of Biosciences, 31(1), 82–93. https://doi.org/10.4308/hjb.31.1.82-93
Scheuermann, K. K. & Pereira, A. (2023). Development of a molecular marker for the Pi1 gene based on the association of the SNAP protocol with the touch up gradient Journal 214, of amplification Microbiological method. Methods, 106845. https://doi.org/10.1016/j.mimet.2023.106845
Sintia, M., Ardie, S. W., & Suwarno, W. B. (2023a). Genetic variability of F2 foxtail millet population derived from ICERI-5 and Botok-10 cross. Biodiversitas, 24(6), 3559–3567. https://doi.org/10.13057/biodiv/d240655
Sintia, M., Suwarno, W. B., & Ardie, S. W. (2023b). Genetic Study of Three F2 Populations of foxtail millet [Setaria italica (L.) Beauv.] Assisted by SiDREB2-based SNAP Marker for Drought and/ or Salinity Tolerance [Master thesis, IPB University]. IPB University Scientific Repository.
Wang, C., Chen, J., Zhi, H., Yang, L., Li, W., Wang, Y., Li, H., Zhao, B., Chen, M., & Diao, X. (2010). Population genetics of foxtail millet and its wild ancestor. BMC Genetics, 11(1), 90. https://doi.org/10.1186/1471-2156-11-90
Widyawan, M. H., Khumaida, N., Kitashiba, H., Nishio, T., & Ardie, S. W. (2018a). Optimization of dot-blot SNP analysis for the detection of drought or salinity stress associated marker in foxtail millet (Setaria italica L.). SABRAO Journal of Breeding and Genetics, 50(1), 72–84.
Widyawan, M. H., Khumaida, N., & Ardie, S. W. (2018b). Development of transcription factor gene-based SNP marker for drought or salinity tolerance in foxtail millet (Setaria italica (L.) Beauv) [Master thesis, IPB University]. IPB University Scientific Repository.
Zhang, J., Li, K., Liao, D., Pardinas, J. R., Chen, L., & Zhang, X. (2003a). Different applications of polymerases with and without proofreading activity in single nucleotide polymorphism Laboratory Investigation, analysis. 83(8),1147 1154. https://doi.org/10.1097/01.LAB.0000081589.91390.DF
Zhang, J., Li, K., Deng, Z., Liao, D., Fang, W., & Zhang, X. (2003b). Efficient mutagenesis method for producing the template of single nucleotide polymorphisms. Molecular Biotechnology, 24, 105-10. https://doi.org/10.1385/MB:24:2:105
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